Methods of gene delivery using capsid-modified raav expression systems

ABSTRACT

Disclosed are methods of gene delivery using capsid-modified recombinant adeno-associated viral (rAAV) vectors. Exemplary methods are provided employing vectors that have altered affinity for heparin or heparin sulfate, as well as vectors, expression systems, and rAAV virions that lack functional VP2 protein expression, but are nevertheless, fully virulent. Also provided by the invention are methods employing the rAAV vector-based compositions, virus particles, host cells, and pharmaceutical formulations in the expression of selected therapeutic proteins, polypeptides, peptides, antisense oligonucleotides and/or ribozymes in selected mammals, including organs, tissues, and human host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 10/513,059, filed Oct. 2, 2005 (allowed; Atty. Dkt. No. 36689.32);which was a U.S. §371 nationalization of PCT Intl. Pat. Appl. No.PCT/US03/13583, filed May 1, 2003 (nationalized); which claims priorityto U.S. Prov. Pat. Appl. No. 60/377,315, filed May 1, 2002 (expired);the contents of each of which is specifically incorporated herein in itsentirety by express reference thereto.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. HL59412and HL51811 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and virology, and in particular, to the development of genedelivery vehicles. The invention provides improved recombinantadeno-associated virus (rAAV) vectors that while deleted for VP2, arestill able to form infectious virion particles, as well as other AAVvector compositions useful in expressing a variety of nucleic acidsegments, including those encoding therapeutic proteins polypeptides,peptides, antisense oligonucleotides, and ribozyme constructs, invarious gene therapy regimens. Methods are also provided for preparingand using these modified rAAV-based vector constructs in a variety ofviral-based gene therapies, and in particular, treatment and preventionof human diseases using conventional gene therapy approaches. Theinvention also provides multicomponent vector systems which may be usedto assess the relative efficiency and infectivity of a variety of AAVparticles having mutated, or deleted capsid proteins.

2. Description of Related Art

Major advances in the field of gene therapy have been achieved by usingviruses to deliver therapeutic genetic material. The adeno-associatedvirus (AAV) has attracted considerable attention as a highly effectiveviral vector for gene therapy due to its low immunogenicity and abilityto effectively transduce non-dividing cells. AAV has been shown toinfect a variety of cell and tissue types by using heparin sulfateproteoglycan (HSPG) as its primary cellular receptor. The naturaltropism of AAV for the abundantly expressed HSPG presents a challenge tospecifically targeting particular cell populations. For safety andtargeting considerations it is highly desirable to have a vector thatcannot infect its natural host cell types.

SUMMARY OF THE INVENTION

The present invention overcomes these and other limitations inherent inthe prior art by providing new rAAV-based genetic constructs that encodeone or more mammalian therapeutic polypeptides for the prevention,treatment, and/or amelioration of various disorders resulting from adeficiency in one or more of such polypeptides. In particular, theinvention provides AAV-based genetic constructs encoding one or moremammalian therapeutic proteins, polypeptides, peptides, antisenseoligonucleotides, and ribozymes, as well as variants, and/or activefragments thereof, for use in the treatment and prophylaxis of a varietyof conditions and mammalian diseases and disorders.

Current AAV2 targeting strategies involve inserting DNA sequences thatcode for specific receptor ligands within the capsid open reading frameof the pIM45 plasmid. While this approach has identified surfacepositions capable of tolerating peptide insertions, there are certainlimitations. Because the three capsid proteins share the same openreading frame and stop codon, the amino acid sequence of the majorcapsid protein, VP3, and any peptide ligands inserted in this region ofthe open reading frame, are contained within the 2 larger andsignificantly less abundant capsid proteins, VP1 and VP2.

In order to target peptide ligands to a specific capsid protein, theinventors have investigated an alternative method for the production ofrecombinant AAV2 vectors. By mutating the capsid proteins' start codonsthe inventors have generated pIM45 plasmids that only express one capsidprotein: pIM45-VP1, pIM45-VP2 (acg/atg), and pIM45-VP3. Such plasmidscan be complemented with plasmids that express the remaining 2 capsidproteins (pIM45-VP2,3, pIM45-VP1,3, and pIM45-VP1,2; respectively) inorder to produce viable recombinant AAV2 vectors. Interestingly, theplasmid, pIM45-VP1,3, is also capable of producing infectious virions inthe absence of VP2 expression. Expression of the capsid proteins in thismanner allows for the genetic modification of a specific capsid proteinacross its entire sequence. As a result, more control of the positionand number of expressed peptide insertions is obtained in producingrecombinant AAV2 vectors. This system allows for the production of noveltargeted recombinant AAV2 vectors containing significantly largerpeptide insertions in an individual capsid protein without disruption ofthe remaining capsid structure.

In one embodiment, the invention concerns rAAV vectors that comprise anucleic acid segment modified to express functional VP1 and VP3 capsidproteins substantially in the absence of functional VP2 protein.Surprisingly, the inventors have shown that such a vector can produce aninfectious virion in the absence of exogenous VP2 protein.

The lack or substantial absence of functional VP2 protein may be theresult of at least a first mutation in the capsid gene sequence regionthat comprises the VP2 start codon, or alternatively in the VP2 startcodon itself. An exemplary vector described herein is pIM45-VP1,3.

In another embodiment, the invention concerns rAAV vectors that comprisea nucleic acid segment modified to express functional VP1 and VP2 capsidproteins substantially in the absence of functional VP3 protein.Although such vector cannot produce an infectious virion in the absenceof exogenous VP3 protein, if a second helper vector that encodes afunctional VP3 protein is employed to coinfect cells with this vector,infectious virions can be obtained.

The lack or substantial absence of functional VP3 protein may be theresult of at least a first mutation in the capsid gene sequence regionthat comprises the VP3 start codon, or alternatively in the VP3 startcodon itself. An exemplary vector described herein is pIM45-VP1,2.

In a third embodiment, the invention concerns rAAV vectors that comprisea nucleic acid segment modified to express functional VP2 and VP3 capsidproteins substantially in the absence of functional VP1 protein.Although such vector cannot produce an infectious virion in the absenceof exogenous VP1 protein, if a second helper vector that encodes afunctional VP1 protein is employed to coinfect cells with this vector,infectious virions can be obtained.

The lack or substantial absence of functional VP1 protein may be theresult of at least a first mutation in the capsid gene sequence regionthat comprises the VP1 start codon, or alternatively in the VP1 startcodon itself. An exemplary vector described herein is pIM45-VP2,3.

A yet further embodiment of the invention is an expression vector thatexpresses an rAAV capsid protein selected from the group consisting ofVP1, VP2, and VP3, each in the absence of substantially any other rAAVprotein, such as the other capsid proteins or helper functions.

This expression vector may comprise, for example, a mutation at position1 of the cap gene, a mutation at position 138 of the cap gene, or amutation at position 203 of the cap gene. Exemplary such vectorsprovided herein are pIM45-VP1, pIM45-VP2, or pIM45-VP3, which producesubstantially a single VP1, VP2, or VP3 protein, respectively.

Another embodiment of the invention is an expression vector thatexpresses: (a) rAAV capsid proteins VP1 and VP2 in the absence ofsubstantial amounts of VP3 protein; (b) rAAV capsid proteins VP1 and VP3in the absence of substantial amounts of VP2 protein; or (c) rAAV capsidproteins VP2 and VP3 in the absence of substantial amounts of VP1protein.

Such vector typically comprises: (a) at least one mutation in the startcodon of the VP1 protein and at least one mutation in the start codon ofthe VP2 protein; (b) at least one mutation in the start codon of the VP1protein and at least one mutation in the start codon of the VP3 protein;or (c) at least one mutation in the start codon of the VP2 protein andat least one mutation in the start codon of the VP3 capsid protein.

For example, the vector may comprise: (a) at least one mutation atposition 1 and at least one mutation at position 138 of the cap gene,(b) at least one mutation at position 1 and at least one mutation atposition 203 of the cap gene; or (c) at least one mutation at position138 and at least one mutation at position 203 of the cap gene. VectorspIM45-VP1,2; pIM45-VP1,3; and pIM45-VP2,3 described herein, arerepresentative examples of each of such vectors, respectively.

The invention also provides in an important embodiment, an rAAVexpression system substantially lacking in expression of VP2 protein.This VP2-free system comprises:(a) at least a first rAAV vectorcomprising at least a first heterologous nucleic acid segment insertedinto the capsid sequence region, with the segment encoding at least afirst heterologous peptide; and (b) at least a second expression vectorthat expresses functional VP1 and VP3 capsid proteins in the absence ofsubstantial quantities of VP2 protein, or at least a second and a thirdexpression vector that separately express functional VP1 and VP3 capsidproteins, each of these second and third plasmids expressing a singleVP1 or VP3 protein, both in the absence of substantial amounts of VP2protein.

For example, the system will preferably comprise at least a first rAAVvector that substantially lacks VP2 expression. Such expression systemswill give rise to infectious virions, so long as the helper plasmidsprovide sufficient exogenous VP1 and VP3 protein to permit the rAAVvector to form the capsid.

In one embodiment, when it is desirable to “target” particular cells,cell surfaces, or cell surface ligands or receptors, it may be desirableto alter the sequence of the capsid gene through the addition of one ormore relatively short nucleic acid segments that encode at least 1 ormore targeting peptides that, when these heterologous peptides areexpressed on the surface of an rAAV virion comprising the vector, thepeptide sequence contained within the altered capsid protein will permitthe selective targeting of the rAAV virions comprising them to one ormore specific types of cells, cell surfaces, or cell surface receptorswhen the particles are used to transfect a plurality or population ofsuch host cells. The inventors contemplate that the exploitation of suchtargeting peptide sequences, when expressed on the surface of the rAAVvirions as contained within the capsid proteins, may be critical inlocalizing, enhancing, improving, or increasing the specificity of therAAV virions for a particular cell type, or may even be useful inpermitting transduction of cells or cell types that previously were notappropriate host cells for AAV infection. Such methods could beparticularly desirable in altering the native affinity of one or more ofthe various known serotypes of AAV to one or more host cells notpreviously capable of efficient transfection by one or more particularserotypes. For example, by appropriate insertion of one or more peptideepitopes, ligands, or recognition sequences, an rAAV serotype 1 vectormay be able to efficiently transfect a cell line not readily transfectedby wild-type rAAV1 vectors. Likewise, an rAAV serotype 2 vector may besufficiently modified by addition of appropriate targeting ligands toeffectively transfect one or more cell lines, cells types, tissues, ororgans, not previously capable of efficient transfection using theunmodified wild-type rAAV2 vector.

As such, preferred embodiments include those VP2-free rAAV expressionsystems, wherein at least a first peptide inserted into one or more ofthe capsid protein sequences, permits the rAAV virion to transfect aspecific organ tissue, or host cell, with a higher efficiency than anunmodified rAAV vector.

The VP2-free rAAV expression systems of the invention may utilize anyrAAV vector, including those of serotypes 1, 2, 3, 4, 5, or 6, and mayemploy at least two helper plasmids such as pIM45-VP1, pIM45-VP2, orpIM45-VP3, as the second and third expression vectors required in thesystem to provide exogenous VP1, VP2, and/or VP3 as may be required forefficient virion formation by the rAAV vectors. When only a secondhelper plasmid is desired, a single vector may be employed such as, forexample, pIM45-VP1,3. Alternatively, so long as at least VP1 and VP3 areprovided to the system, either on a single plasmid, each on separateplasmids, or by exogenous supplementation of one or both of the purifiedprotein(s) themselves, a fully functional, fully virulent rAAV virionmay be reconstituted from the disclosed expression system, either in thepresence of functional VP2 protein, or alternatively, substantially inthe absence of any endogenously- or exogenously-provided VP2 protein.

When the use of such vectors is contemplated for introduction of one ormore exogenous proteins, polypeptides, peptides, ribozymes, and/orantisense oligonucleotides, to a particular cell transfected with thevector, one may employ the rAAV vectors or the VP2-free rAAV expressionsystems disclosed herein by genetically modifying the vectors to furthercomprise at least a first exogenous polynucleotide operably positioneddownstream and under the control of at least a first heterologouspromoter that expresses the polynucleotide in a cell comprising thevector to produce the encoded peptide, protein, polypeptide, ribozyme,or antisense oligonucleotide. Such constructs may employ heterologouspromoters that are constitutive, inducible, or even cell-specificpromoters. Exemplary such promoters include, but are not limited to, aCMV promoter, a β-actin promoter, a hybrid CMV promoter, a hybridβ-actin promoter, an EF1 promoter, a U1a promoter, a U1b promoter, aTet-inducible promoter and a VP16-LexA promoter.

The vectors or expression systems may also further comprise one or moreenhancers, regulatory elements, transcriptional elements, to alter oreffect transcription of the heterologous gene cloned in the rAAVvectors. For example, the rAAV vectors of the present invention mayfurther comprise at least a first CMV enhancer, a synthetic enhancer, ora cell- or tissue-specific enhancer. The exogenous polynucleotide mayalso further comprise one or more intron sequences.

In other aspects, the invention concerns methods for altering, reducing,or eliminating, the binding of particular rAAV vectors for particularligands. In an illustrative embodiment, the invention provides rAAVvectors that have altered affinity for heparin, heparin sulfate, andheparin sulfate proteoglycan. This vector comprises at least a firstmutation in the capsid gene, wherein the mutation substantially reducesor eliminates the affinity of a viral particle comprising the vector forbinding to heparin, heparin sulfate, or heparin sulfate proteoglycan.Preferably, these rAAV vectors comprise one or more Arginine to Alaninemutations, and particularly one or more Arginine to Alanine mutations atposition 585 or position 588 of the capsid polypeptide sequence. In rAAVvectors comprising either a single R585A or R588A mutation, or a doublemutant comprising both the R585A and the R588A mutations, affinity forheparin sulfate binding by the vector was eliminated. Such vectors aretherefore important when one wishes to design improved rAAV vectors thatcomprise particular capsid protein mutations that either have increasedor reduced affinity for one or more particular ligands.

In all aspects of the invention, the exogenous polynucleotides that arecomprised within one or more of the improved rAAV vectors disclosedherein will be of mammalian origin, with human, murine, porcine, bovine,ovine, feline, canine, equine, epine, caprine, and lupinepolynucleotides being particularly preferred.

As described above, the exogenous polynucleotide will preferably encodeone or more proteins, polypeptides, peptides, ribozymes, or antisenseoligonucleotides, or a combination of these. In fact, the exogenouspolynucleotide may encode two or more such molecules, or a plurality ofsuch molecules as may be desired. When combinational gene therapies aredesired, two or more different molecules may be produced from a singlerAAV expression system, or alternatively, a selected host cell may betransfected with two or more unique rAAV expression systems, each ofwhich will provide unique heterologous polynucleotides encoding at leasttwo different such molecules.

In other embodiment, the invention also concerns the disclosed rAAVvectors comprised within an infectious adeno-associated viral particle,comprised within one or more pharmaceutical vehicles, and may beformulated for administration to a mammal such as a human fortherapeutic, and/or prophylactic gene therapy regimens. Such vectors mayalso be provided in pharmaceutical formulations that are acceptable forveterinary administration to selected livestock, domesticated animals,pets, and the like.

The invention also concerns host cells that comprise the disclosed rAAVvectors and expression systems, particularly mammalian host cells, withhuman host cells being particularly preferred.

Compositions comprising one or more of the disclosed rAAV vectors,expression systems, infectious AAV particles, host cells also form partof the present invention, and particularly those compositions thatfurther comprise at least a first pharmaceutically-acceptable excipientfor use in the manufacture of medicaments and methods involvingtherapeutic administration of such rAAV vectors. Such pharmaceuticalcompositions may optionally further comprise liposomes, a lipid, a lipidcomplex; or the rAAV vectors may be comprised within a microsphere or ananoparticle. Pharmaceutical formulations suitable for intramuscular,intravenous, or direct injection into an organ or tissue of a human areparticularly preferred.

Other aspects of the invention concern recombinant adeno-associatedvirus virion particles, compositions, and host cells that comprise oneor more of the AAV vectors disclosed herein, such as for examplepharmaceutical formulations of the vectors intended for administrationto a mammal through suitable means, such as, by intramuscular,intravenous, or direct injection to cells, tissues, or organs of aselected mammal. Typically, such compositions may be formulated withpharmaceutically-acceptable excipients as described hereinbelow, and maycomprise one or more liposomes, lipids, lipid complexes, microspheres ornanoparticle formulations to facilitate administration to the selectedorgans, tissues, and cells for which therapy is desired.

Kits comprising one or more of the disclosed vectors, virions, hostcells, viral particles or compositions; and (ii) instructions for usingthe kit in therapeutic, diagnostic, or clinical embodiments alsorepresent preferred aspects of the present disclosure. Such kits mayfurther comprise one or more reagents, restriction enzymes, peptides,therapeutics, pharmaceutical compounds, or means for delivery of thecompositions to host cells, or to an animal, such as syringes,injectables, and the like. Such kits may be therapeutic kits fortreating or ameliorating the symptoms of particular diseases, and willtypically comprise one or more of the modified AAV vector constructs,expression systems, virion particles, or therapeutic compositionsdescribed herein, and instructions for using the kit.

Another important aspect of the present invention concerns methods ofuse of the disclosed vectors, virions, expression systems, compositions,and host cells described herein in the preparation of medicaments fortreating or ameliorating the symptoms of various polypeptidedeficiencies in a mammal. Such methods generally involve administrationto a mammal, or human in need thereof, one or more of the disclosedvectors, virions, host cells, or compositions, in an amount and for atime sufficient to treat or ameliorate the symptoms of such a deficiencyin the affected mammal. The methods may also encompass prophylactictreatment of animals suspected of having such conditions, oradministration of such compositions to those animals at risk fordeveloping such conditions either following diagnosis, or prior to theonset of symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thefollowing description taken in conjunction with the accompanyingdrawings, in which like reference numerals identify like elements, andin which:

FIG. 1A-1, FIG. 1A-2, FIG. 1B-1, FIG. 1B-2, FIG. 1C-1, and FIG. 1C-2show generation of plasmids that express two capsid proteins throughmissense mutation of individual capsid protein start codons. FIG. 1Ashows mutations required to eliminate VP1 and VP2 expression. Immunoblotof whole cell lysates using B1 antibody that recognizes all threecapsids following transfection of plasmids, pIM45(lane 1); pIM45-VP2,3(lane 2); pIM45-VP1,3 (lane 3); and pIM45-M203L (lane 4). Note: lane 4is the initial attempt to produce plasmid that expresses only VP1 andVP2. Further mutations are required. FIG. 1B-1 and FIG. 1B-2 showmutations required to eliminate VP3 expression. Immunoblot of whole celllysates using B1 antibody that recognizes all three capsid followingtransfection of pIM45 (lane 1); pIM45-M203L (lane 2); pIM45-M203,211L(lane 3); pIM45-M203,211,235L (lane 4). Note, pIM45-M203,211,235L isdesignated pIM45-VP1,2. FIG. 1C-1 and FIG. 1C-2 show alternativemutation used to eliminate VP3 expression while maximizing expression ofVP2 protein. Immunoblot of whole cell lysates using B1 antibody thatrecognizes all three capsid proteins following transfection of pIM45(lane 1) and pIM45-VP1,2A (lane 3) in which the start codon for VP2protein is changed from ACG to ATG.

FIG. 2A and FIG. 2B show generation of plasmids that express a singlecapsid protein. Immunoblot of whole cell lysates using B1 antibody thatrecognizes all three capsid proteins following transfection of pIM45(lane 1); pIM45-VP1 (lane 2); pIM45-VP2 (lane 3) pIM45-VP2A (lane 4);and pIM45-VP3 (lane 5);

FIG. 3A-1, FIG. 3A-2, FIG. 3B-1, FIG. 3B-2, FIG. 3C-1 and FIG. 3C-2 showproduction and purification of rAAV2-like particles that lack expressionof specific capsid proteins. FIG. 3A-1 and FIG. 3A-2 show analysis ofeffects of missense mutations required to eliminate VP3 expression. FIG.3A-1 shows immunoblot using B1 antibody that recognizes all three capsidproteins of purified particle stocks from pIM45 (lane 1); pIM45-M203L(lane 2); pIM45-M211L (lane 3); pIM45-M235L (lane 4), pIM45-M203,211,235(lane 5). FIG. 3A-2 shows dot blot autoradiograph of DNA extracted fromsame particle stocks. Aliquots from an iodixinal step gradient were withincubated with DNAseI, inactivated with EDTA, digested with proteinaseK, phenol:chloroform extracted, and precipitated with ethanol. DNA wastransferred to nitrocellulose and probed with radiolabelled GFP probe.FIG. 3B-1 and FIG. 3B-2 show analysis of effects of eliminating a singlecapsid on the production and purification of virus particles. FIG. 3B-1shows immunoblot using B1 antibody that recognizes all three capsidproteins of purified particle stocks from pIM45 (lane 1); pIM45-VP1,2(lane 2); pIM45-VP1,3 (lane 3); and pIM45-VP2,3 (lane 4). FIG. 3B-2shows dot blot autoradiograph of DNA extracted from same particlestocks. Aliquots from an iodixinal step gradient were with incubatedwith DNAse I, inactivated with EDTA, digested with proteinase K,phenol:chloroform extracted, and precipitated with ethanol. DNA wastransferred to nitrocellulose and probed with radiolabelled GFP probe(FIG. 3C-1 and FIG. 3C-2);

FIG. 4 shows complementation capsid plasmid groups employed to produceviable rAAV2 particle preparations. Group VP0 is a control groupconsisting of pIM45 and pIM45-VP0 (all capsid expression eliminated).Group VP1 is group consisting of pIM45-VP1 and pIM45-VP2,3 in whichexpression of VP1 is isolated. Group VP2/VP2A is group consisting ofpIM45-VP2 or pIM45-VP2A and pIM45-VP1,3 in which expression of VP2 isisolated, and in case of pIM45-VP2A, VP2 expression is maximized. GroupVP3 is group consisting of pIM45-VP3 and pIM45-VP1,2 in which expressionof VP3 is isolated. Isolation of specific capsid proteins allows geneticmodification of the isolated capsid without further modifying remainingcapsids. Alternatively, genetic modification of two capsids can beaccomplished without further modification of remaining capsid. Thesegroups are cotransfected with pXX6 (Ad helper functions) and pTR-UF5(terminal repeats flanking expression cassette with CMV promoter drivingexpression of GFP) to produce rAAV vectors;

FIG. 5A-1, FIG. 5A-2, FIG. 5B-1 and FIG. 5B-2 show production andpurification of rAAV2-like particles from complementation groupsdescribed in FIG. 4. FIG. 5A-1 shows immunoblot using B1 antibody thatrecognizes all three capsid proteins of purified particle stocks fromGroup VP0(lane 1); Group VP1 (lane 2); Group VP2 (lane 3); Group VP2A(lane 4); and Group VP3 (lane 5). Note lane 4 shows production ofparticle stock with increased level of VP2 protein in resultantparticles composed of all three capsid proteins. FIG. 5A-2 shows dotblot autoradiograph of DNA extracted from same particle stocks. Aliquotsfrom an iodixinal step gradient were with incubated with DNAse I,inactivated with EDTA, digested with proteinase K, phenol:chloroformextracted, and precipitated with ethanol. DNA was transferred tonitrocellulose and probed with radiolabelled GFP probe. FIG. 5B-1 showsimmunoblot using B1 antibody that recognizes all three capsid proteinsof purified particle stocks from transfection of pIM45-VP2A andpIM45-VP3 showing production of rAAV2-like particles composed of VP2 andVP3 with increased VP2 levels relative to VP3. FIG. 5B-2 shows dot blotautoradiograph of DNA extracted from same particle stocks. Aliquots froman iodixinal step gradient were with incubated with DNAseI, inactivatedwith EDTA, digested with proteinase K, phenol:chloroform extracted, andprecipitated with ethanol. DNA was transferred to nitrocellulose andprobed with radiolabelled GFP probe;

FIG. 6A, FIG. 6B-1, FIG. 6B-2, FIG. 6C-1, and FIG. 6C-2 depictproduction of rAAV2-like particles with large peptide insertions in VP1and VP2 capsid proteins. FIG. 6A shows production scheme for insertionof large peptides in VP1 and VP2 (top) involves insertion of peptideimmediately after amino acid 138 in a plasmid that expresses only VP1and VP2 (pIM45-VP1,2A) and complementing this plasmid with plasmid,pIM45-VP3, to produce particles. Production scheme for insertion oflarge peptides only in VP2 (bottom) involves insertion of peptideimmediately after amino acid 138 in a plasmid that expresses only VP2(pIM45-VP2A) and complementing this plasmid with plasmid, pIM45-VP1,3 toproduce particles. FIG. 6B-1 and FIG. 6B-2 show immunoblot of purifiedrAAV2-like particles produced by above production schemes with protein,leptin, inserted in VP1 and VP2 or only in VP2. FIG. 6B-1 showsimmunoblot probed with antibody recognizing all three capsids proteins.FIG. 6B-2 shows immunoblot probed with antibody recognizing insertedpeptide, leptin. Both panels: Lane 1: pIM45; Lane 2:pIM45-VP1,2A-Leptin/pIM45-VP3; Lane 3: pIM45-VP2A-Leptin/pIM45-VP1,3;Lane 4: pIM45-VP3 only; and Lane 5: pIM45-VP1,3 only. FIG. 6C-1 and FIG.6C-2 show immunoblot of purified rAAV2-like particles produced by aboveproduction schemes with protein, GFP, inserted in VP1 and VP2 or only inVP2. FIG. 6C-1 shows immunoblot probed with antibody recognizing allthree capsids proteins. FIG. 6C-2 shows immunoblot probed with antibodyrecognizing inserted peptide, GFP. Both panels: Lane 1: pIM45; Lane 2:pIM45-VP1,2A-GFP/pIM45-VP3; Lane 3: pIM45-VP2A-GFP/pIM45-VP1,3; Lane 4:pIM45-VP3 only; and Lane 5: pIM45-VP1,3 only;

FIG. 7 shows Western blot of iodixanol virus stocks. Equal volumes ofvirus stock were separated by 10% SDS-PAGE and analyzed by Western blotusing the B1 antibody;

FIG. 8 shows heparin-agarose binding profiles of mutant capsids.Approximately 5×10¹⁰ particles were applied to 500 μL of heparin-agaroseaffinity matrix at a NaCl concentration of 100 mM, washed extensivelywith the loading buffer, and bound capsids were eluted with 2 M NaCl.Pooled fractions were denatured and slot blotted onto nitrocellulose forimmunodetection with mAb B1. For each mutant, L is the total amount ofiodixanol purified virus that was loaded onto the heparin agarosecolumn; FT is the total virus that flowed through the column, W is thewash; E is the eluate;

FIG. 9A and FIG. 9B show production and purification of AAV serotypes.FIG. 9A shows equivalent amounts of iodixanol purified AAV1, AAV2 andAAV5 were separated by 10% PAGE and analyzed by Western blot using theB1 antibody. FIG. 9B shows heparin-agarose binding properties of AAV2,AAV1 and AAV5. Abbreviations are the same as FIG. 8;

FIG. 10 shows particle-to-infectivity ratios of mutants relative to wildtype. The particle-to-infectivity ratio for each mutant was calculatedby dividing the average genomic titer by the average green cell assaytiter (Table 2). The P/I ratio of each mutant was then normalized towild type by dividing the P/I of each mutant by the P/I of wild typerAAV2, and the log₁₀ value of the ratio was plotted. Wild type,therefore, equals one, and is indicated by the dashed line. Hatchedbars, mutant viruses with infectivity comparable to wild type; graybars, mutant viruses that are heparin binding deficient; White bars,mutant viruses with an undetermined block to infectivity; Asterisksindicate those mutants for which no green cells were scored. For thesemutants the green cell assay titer used was the limit of detection inthe assay. Thus, the log difference is a minimum estimate.

FIG. 11 shows GFP transduction ability of mutants in HeLa C12 cells.Cells were infected with wild type rAAV or mutant virus at an MOI=500genomic particles and an Ad5 MOI=10 pfu per cell. Twenty-four hrs postinfection cells were fixed with 2% paraformaldehyde and the number ofGFP positive cells was determined by FACS analysis;

FIG. 12A and FIG. 12B show binding and uptake of rAAV2 and R585A/R588Agenomes in Hela C12 cells. FIG. 12A shows 10⁶ cells were infected withrAAV2 or R585/R588A at an M01=100 or 1000 genome containing particlesper cell, respectively. At the indicated times, infection media wasremoved and saved. The cells were washed and harvested, and Hirt DNA wasextracted from both the infection media and the cell pellet. Southernanalysis was performed using an [α-³²P]-dATP labeled GFP probe. FIG. 12Bshows the percent bound/internalized DNA was calculated by dividing thetotal DNA present in both the media and the cell pellet by the amountbound/internalized for each time point. The average of threedeterminations is shown. Error bars indicate a standard deviation;

FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D show modifying the heparinbinding properties of AAV5. FIG. 13A shows alignment of AAV2 amino acidresidues 585 through 590 (RGNRQA; SEQ ID NO:1) to residues predicted byamino acid alignment to be structurally equivalent in AAV5 (SNSNLP; SEQID NO:2). FIG. 13B shows Western blot of iodixanol virus stocks. Equalvolumes of virus were separated by 10% SDS-PAGE and analyzed by Westernblot using the B1 antibody. FIG. 13C shows novel heparin bindingproperties of AAV5-HS. Heparin-agarose binding was performed asdescribed in FIG. 8. See FIG. 8 for abbreviations. FIG. 13D shows thelog of the particle-to-infectivity ratio of the rAAV5 variantsnormalized to wild type rAAV2 as described in FIG. 10;

FIG. 14 shows an immunoslotblot of total capsid protein from novelproduction system following standard purification procedures.Immunoslotblot was probed with anti-VP1,2,3 monoclonal antibody. Lane 1:pIM45/pIM45-VPO; Lane 2: pIM45-VP1/pIM45-VP2,3; Lane 3:pIM45-VP2acg/pIM45-VP1,3; Lane 4: pIM45-VP2atg/pIM45-VP1,3; and Lane 5:pIM45-VP3/pIM45-VP1,2;

FIG. 15 shows a dot blot autoradiograph of DNA extracted from pTR-UF5and system plasmid combinations. Numbering scheme is the same asdescribed in FIG. 14. Equal volume aliquots from an iodixinol stepgradient were with incubated with DNAseI, inactivated with EDTA,digested with proteinase K, phenol:chloroform extracted, andprecipitated with ethanol. DNA was transferred to nitrocellulose andprobed with radiolabeled GFP probe;

FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D show the in vivo transductionability of recombinant AAV vectors produced using various systemcomponents. GFP fluorescence microscopy was performed on Hela C12infected at an MOI of 1000 genomes/cell 24 hrs post infection.

FIG. 17A and FIG. 17B show the Immunoblot and dot blot autoradiograph ofvirions produced from pTR-UF5; pIM45-VP1,2; pIM45-VP1,3; and pIM45-VP2,3plasmids following standard purification protocols. The capsid proteinsVP1, VP2, and VP3 are indicated. No virions were obtained in 40%iodixanol fraction from plasmid pIM45-VP1,2;

FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show the in vivo transductionability of recombinant AAV vectors containing only two capsid proteins.GFP fluorescence microscopy was performed on Hela C12/24 hrs postinfection;

FIG. 19 depicts an immunoblot of protein fractions collected fromiodixinol purified passed over a heparin-agarose column. Immunoblot wasprobed with anti-VP1,2,3 monoclonal antibody. C: 5¹⁰ virus particlesloaded directly onto blot, FT: flowthrough fraction, W: wash fraction,and E: 2 M NaCl fraction;

FIG. 20 shows a dot blot autoradiograph of DNA extracted from pTR-UF5and rAAV R585A, R588A. Equal volume aliquots from an iodixinal stepgradient were with incubated with DNAseI, inactivated with EDTA,digested with proteinase K, phenol:chloroform extracted, andprecipitated with ethanol. DNA was transferred to nitrocellulose andprobed with radiolabeled GFP probe;

FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21D show the in vivo transductionability of pTR-UF5 and R585A, R588A. GFP fluorescence microscopy wasperformed on Hela C12 and HEK 293 cells infected at an MOI of 1000genomes/cell 24 hrs post infection;

FIG. 22 shows a slot blot autoradiograph of an in vivo DNA trackingtime-course experiment of pTR-UF5, rAAV R585A, R588A. Media and cellsinfected with pTR-UF5 and rAAV R585A, R588A were collected at 1, 4, and20 hrs post infection. Hirt DNA was extracted, transferred tonitrocellulose and probed with a radiolabeled GFP probe; and

FIG. 23 shows a schematic diagram of the pIM45 vector showing the repand cap sequences.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Illustrative embodiments of the invention are described below. In theinterest of clarity, not all features of an actual implementation aredescribed in this specification. It will of course be appreciated thatin the development of any such actual embodiment, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which will vary from one implementation toanother. Moreover, it will be appreciated that such a development effortmight be complex and time-consuming, but would nevertheless be a routineundertaking for those of ordinary skill in the art having the benefit ofthis disclosure.

rAAV Type 2

The adeno-associated virus type 2 (AAV2) is a small, non-envelopedparvovirus that has received considerable attention as a gene therapyvector (see, e.g., Muzyczka and Berns, 2001). The capsid has a diameterof approximately 20 nm formed by an icosahedral lattice with T=1symmetry (60 structurally equivalent subunits). In purified virions,three structural proteins, VP1, VP2, and VP3 with molecular masses of87, 73, and 62 Kda, respectively, are present in a molar ratio of 1:1:18(Buller and Rose, 1978). mRNAs encoding capsid proteins are synthesizedfrom a single open reading frame and use alternative splicing and startcodons to produce three VP proteins that share an identical 532carboxyl-terminal amino acid domain (Becerra et al., 1988; Becerra etal., 1985), with VP2 and VP3 containing successive amino terminaltruncations of VP1.

The atomic structure of the AAV2 capsid has been determined to aresolution of 3.5 angstroms (Xie et al., 2002). In this model, sixtycopies of VP3 minus 14 amino terminal residues are present in anicosahedral arrangement. The VP3 protein contains 8 anti-parallelβ-strands that adopt a barreloid structure similar to capsid proteins ofother non-enveloped viruses. Loops of variable length connect theinterior β-barrel scaffold and extend outwards to form the capsidsurface. Cryo-electron microscopy of empty AAV2 particles generated asurface density map that described holes, spikes and canyon featuressimilar to those found in other parvoviruses (Kronenberg et al., 2001).Before the crystal structure was available, several alternative methodswere investigated in an attempt to localize specific regions of thecapsid. Neutralizing antibody screening of peptide sequences derivedfrom VP1 found multiple antigenic determinants distributed on the capsidexterior in both linear and conformation dependent forms (Moskalenko etal., 2000). Computer modeling of AAV structure based on the known atomicstructure of the related canine parvovirus coupled with geneticmodification of the capsid identified several positions that were on thesurface of the capsid and could tolerate insertions and substitutions(Grifman et al., 2001; Nicklin et al., 2001; Rabinowitz et al., 1999;Ried et al., 2002; Shi et al., 2001; Wu et al., 2000; Yang et al.,1998).

Cell membrane binding and entry initiates all viral infections.Non-enveloped viruses rely on membrane bound extracellular receptors forattachment to the cell membrane. AAV2 has evolved a dynamic andmultistep infectious entry pathway that utilizes the abundantlyexpressed heparan sulfate proteoglycan (HSPG) as its primary target(Summerford and Samulski, 1998). Two co-receptors, αVβ5 integrin andbasic fibroblast growth factor receptor (bFGFR) have been identified,which act as secondary receptors that may stabilize virus attachment orparticipate during internalization (Duan et al., 1999; Qing et al.,1999; Summerford et al., 1999). HSPG is a macromolecule expressed bymany cell types and is a component of the extracellular matrix of mosttissues (see, e.g., Hileman et al., 1998; Mull9oy and Linhardt, 2001).Attached to the core protein are glycosaminoglycan side chains heparinand heparin sulfate (HS). These carbohydrate polymers are formed bydisaccharide repeats consisting of alternating N-acetylglucosamine andiduronic acid residues in a α1,4 linkage. The saccharides can bemodified by N-sulfation as well as 2-O and 6-O-sulfation to impart adense overall negative charge at physiological pH. As a result, HSinteracts with an extensive range of proteins primarily by electrostaticattraction between the electron dense sulfate groups and a cluster ofpositively charged amino acids. Two linear consensus-binding sequences,XBBXBX and XBBBXXBX, and a conformation dependent sequence,TXXBXXTBXXXTBB, (where B is any basic amino acids including His, Lys orArg and X is any hydropathic amino acid and T is a turn) have beenreported (Hileman et al., 1998). Although HSPG is thought to participatein attachment during the infectious process of numerous human viruses(Liu and Thorp, 2002), information about the molecular mechanisms ofthese interactions is limited. A report describing the atomic structureof the foot and mouth disease virus co-crystallized with a HSpentasaccharide is available and serves as the only model defined at theatomic level that describes the molecular interaction between anon-enveloped icosahedral virus and HS (Fry et al., 1999).

Several laboratories have attempted to retarget AAV vectors tonon-permissive cell types by inserting sequences coding for shortforeign peptides into VP3. Interestingly, insertions at position 587,including an L14 integrin binding peptide, a myc tag, an IgG bindingdomain truncation of protein A and an endothelial cell targetingpeptide, abolished the natural heparin binding ability of virus capsidswith these alterations (Girod et al., 1999; Grifman et al., 2001;Nicklin et al., 2001; Ried et al., 2002; Shi et al., 2001). Similarly,an alanine repeat insertion at position 509, an L14 peptide insertion atposition 520, a hemagluttinin tag insertion at positions 522 and 591,and peptides derived from the human luteinizng hormone receptor and thebovine papilloma virus at inserted positions 520 and 584, respectively,have been reported to disrupt heparin binding (Shi et al., 2001; Wu etal., 2000). Curiously, alanine substitutions of acidic residues between561 and 565 also reduced heparin binding, suggesting that nearby basicresidues were affected (Wu et al., 2000). Finally, a substitutionmutation of two arginines and a glutamine at positions 585, 588, and587, respectively, binds poorly to heparin-agarose (Wu et al., 2000).Taken together, these genetic modifications suggested two potentialheparin-binding loci that cluster between positions 509-522 and 561-591.

rAAV Capsid Proteins

Supramolecular assembly of 60 individual capsid protein subunits into anon-enveloped, T-1 icosahedral lattice capable of protecting a 4.7 kbsingle-stranded DNA genome is a critical step in the life-cycle of thehelper-dependent human parvovirus, adeno-associated virus2 (AAV2). Themature 20 nm diameter AAV2 particle is composed of three structuralproteins designated VP1, VP2, and VP3 (molecular masses of 87, 73, and62 kDa respectively) in a ratio of 1:1:18. Based on its symmetry andthese molecular weight estimates, of the 60 capsid proteins comprisingthe particle, three are VP1 proteins, three are VP2 proteins, andfifty-four are VP3 proteins. The employment of three structural proteinsmakes AAV serotypes unique among parvoviruses, as all others knownpackage their genomes within icosahedral particles composed of only twocapsid proteins. The anti-parallel β-strand barreloid arrangement ofthese 60 capsid proteins results in a particle with a defined tropismthat is highly resistant to degradation.

The AAV2 genome contains two large open reading frames (ORF), rep andcap, flanked by inverted terminal repeats. The AAV2 capsid proteins areproduced in an overlapping fashion from the cap ORF; arising throughalternative mRNA splicing of the transcript (initiated at the p40promoter), with subsequent alternative translational start codon usage.A common stop codon is employed for all three capsid proteins. Correctcapsid protein stoichiometry is maintained by translating VP1 from the2.4 KB mRNA, while VP2 and VP3 arise from the 2.3-kB mRNA using a weakerACG start codon for VP2 protein production with resultant read-throughtranslation for the production of the VP3 protein. Differing only in thelength of their N-terminus, these proteins are produced such that theamino acid sequence of VP3 is contained within the significantly lessabundant and longer VP1 and VP2 proteins. As such, VP1's unique 137amino acid N-terminal extension of VP2 contains a phospholipaseenzymatic activity important for viral infectivity. Similarly, VP2extends the N-terminus of VP3 by 64 amino acids with this VP1/VP2overlap region possessing a putative nuclear localization signal (NLS)involved in the nuclear translocation of VP2. The VP3 region common toall three capsid proteins contains the critical β-barrel structuralmotifs characteristic of all parvoviruses and particle surface loopsinvolved in determining viral tropism.

While specific activities have been attributed to regions of anindividual AAV2 capsid protein, the role of each capsid protein in thestructural formation of the particle is less clear. Early studies inwhich all AAV2 capsid expression was eliminated revealed that capsidprotein expression is required for the accumulation of single strandedgenomes. It follows that AAV2 particle assembly occurs within thenucleus and a putative NLS for VP2 has been localized to the VP1/VP2overlap region in transfected COS cells.

In the absence of VP1 expression, this study suggested a major role ofVP2 is the nuclear localization of VP3. However, since VP1 was deletedin this study, one cannot rule out that VP1 has the ability to nuclearlocalize VP3. Site-directed missense mutagenesis of the individualcapsid proteins' start codons suggested that infectious particles areobtained only when all three capsid proteins are present. In contrast,later genetic analysis demonstrated that in the absence of VP1, VP2 andVP3 are able to encapsidate progeny genomes. Similarly, in vitroassembly of purified individual AAV capsid proteins demonstrated thatVP2 and VP3 could form an AAV2-like particle. Baculovirus expression ofthe AAV2 capsid proteins within SF9 cells suggests an absoluterequirement for VP2, although this study failed to eliminate VP3-likefragments produced by the VP2-baculovirus. However, it is feasible thatstudies of AAV2 assembly in baculovirus have subtle differences withparticles assembled in mammalian cells.

An examination of the assembly process of the related autonomous canineparvovirus, CPV, in baculovirus observed significantly more aggregationof capsid proteins in insect cells. In addition, the results of thebaculovirus and NLS studies have the caveat that they were performed inthe absence of AAV2 Rep proteins, Ad helper gene functions, and areplicating AAV2 genome. Furthermore, the p40 promoter in these studiesdoes not control AAV2 capsid protein expression, resulting in alteredstoichiometry of the available capsid protein pool. Indeed, the aboveconcerns seem warranted, as a recent insertional mutagenesis study ofthe AAV2 cap ORF, using standard AAV2 production protocols, reported thepurification of an AAV2-like particle composed of only the VP3 protein.Therefore, despite the uncertainty of the precise role of VP1 and VP2 inparticle formation, the evidence thus far suggests that the VP3 proteinis absolutely required for the formation of an AAV2 particle. Finally,co localization studies of AAV2 assembly in 293 cells demonstrated aninteraction of AAV2 Rep and capsid proteins with Ad proteins and thereplicating genome in the nucleus, thus, supporting a current model ofAAV2 assembly which proposes nucleoplasmic formation of empty particleswith subsequent maturation of the particle as a result of Rep 52/40mediated translocation of capsid protein associated single strandedgenomes into the preformed particles.

Genetic Modification of rAAV Capsid Proteins

Great interest in the assembly, structure, and mutability of the AAV2particle results from its promise as a recombinant gene delivery vehicle(rAAV2) in vivo. Essential to the clinical development of rAAV2 vectorsfor gene therapy is the ability to target specific tissue types.Manipulation of the rAAV2 particle in order to control its cellularreceptor interactions is essential for vector targeting. The feasibilityof various targeting strategies based on AAV cap ORF mutagenesis iscurrently an area of active investigation. A better understanding of theAAV2 particle surface architecture through systematic scanning-alanineand insertional mutagenesis of the AAV cap ORF and recent publication ofthe AAV2 crystal structure has identified several amino acid regions onthe surface of the particle that tolerate sequence alteration withoutloss of capsid stability or integrity.

However, small changes in charge, sequence, and/or position of themutation can result in dramatic changes in the mutant particlephenotype. One limitation in sequence mutation of the overlapping capORF is that mutation of only one capsid protein across its entiresequence is currently not possible. The full potential in manipulationof the particle is not reached with direct alteration of regions ofcapsid overlap. Predicted surface regions of capsid overlap leading todefective phenotypes upon mutagenesis may allow production of viableparticles if such mutations were only in one or two of the capsidproteins. An additional degree of flexibility in modifying the rAAV2particle would result from the ability to mutate the entire codingregion of a specific capsid protein without altering the remaining twocapsid proteins. Indeed, while mutations in the C-terminus of the VP3region have been reported to be completely defective in particleformation following insertion of HA and 6×His tags into the overlappingcap ORF, a recent report focusing on the purification of rAAV2 particlesdemonstrated that the C-terminus of VP3 is capable of accepting a 6×Histag if VP1 and VP2 are not altered. This rAAV2 production strategyinvolved expressing VP1 and VP2 from one construct, and expressing theVP3-6×His fusion protein from a CMV promoter in a second plasmid. In theabsence of the isolation of a specific capsid protein's expression, theN-terminal 137 amino acids of VP1 are the only region of the cap ORFwhere mutations are restricted to a single capsid protein. Successfulinsertions within this region have included HA and serpin. The VP1/VP2overlap region (amino acid 138-202) also has been receptive to sequencemodification. Insertions in this region have included HA, serpin andluetinizing hormone receptor ligand sequences immediately followingamino acid 138 in the cap ORF.

The success of inserting sequences to the VP1 and VP1/VP2 regions may bedue in part to less disruption of the integrity of the particle comparedto insertion in the VP3 region of capsid overlap (amino acid 203-735).It is important to note that these mutant particles would requirefurther mutation of the putative heparin-binding motif to restrictinfection to the target cell. Not surprisingly, since it is the longestregion of capsid protein overlap, contains many critical structuralmotifs, and targeting sequences in this region have 60 representativesin the rAAV particle, mutations in the VP3 region of the AAV2 cap ORFhave resulted in the highest number of defective phenotypes. Yet, onelocation within the VP3 region has received much attention for thesuccessful insertion of small targeting sequences in all three capsidproteins (amino acid 587). One major advantage of targeting insertionsto this position is that the resultant mutant particle also has lost theability to bind its native receptor. Viable mutations in the VP3 regionof the cap ORF have been restricted in size (<30 amino acids).

One caveat of creating genetically-targeted rAAV2 particles, is theconsideration that many cell surface receptors have ligands whose codingsequence are much larger than those successfully inserted directly intothe overlapping cap ORF. Due to the modest size of this ORF (˜2 kB), theinsertion of larger peptide sequences into the capsid coding sequencesmay result in serious disruption of splicing, read-through translation,capsid structure and/or stability. The insertion of large sequences intothe rAAV2 particle have been limited to a study involving the fusion ofthe CD34 single chain antibody coding sequence with the N-termini of theindividual capsid proteins following isolation of their expression toseparate CMV promoters. Viable CD34-retargeted rAAV particles ofextremely low titer were produced only when this fusion was to VP2protein, and co-expression of wild-type VP2 protein was required.Nonetheless, the fusion of large peptide sequences to the N-terminus ofVP2 does not interfere with the incorporation of this capsid proteininto the rAAV2 particle.

Wild-Type AAV2 Binds to Heparan Sulfate Proteoglycan

The adeno-associated virus type-2 (AAV2) uses heparan sulfateproteoglycan (HSPG) as its primary cellular receptor. In order toidentify amino acids within the capsid of AAV2 that contribute to HSPGassociation, biochemical information about heparin/heparin sulfate (HS),AAV serotype protein sequence alignments, and data from previous capsidstudies was used to select residues for mutagenesis. In the presentinvention, charged-to-alanine substitution mutagenesis was performed onindividual and combinations of basic residues for the production andpurification of recombinant viruses that contained a GFP reporter genecassette. Intact capsids were assayed for their ability to bind toheparin-agarose in vitro and virions that packaged DNA were assayed fortheir ability to transduce normally permissive cell lines. It was foundthat mutation of arginine residues at position 585 or 588 eliminatedbinding to heparin-agarose. Mutation of residues R484, R487, and K532showed partial binding to heparin-agarose. A general correlation betweenheparin-agarose binding and infectivity was observed as measured by GFPtransduction; however, a subset of mutants that partially boundheparin-agarose (R484A and K532A) was completely non-infectious,suggesting that they had additional blocks to infectivity that wereunrelated to heparin binding. Conservative mutation of positions R585and R588 to lysine slightly reduced heparin-agarose binding, and hadcomparable effects on infectivity. Substitution of AAV2 residues 585through 590 into a location predicted to be structurally equivalent inAAV5 generated a hybrid virus that bound to heparin-agarose efficiently,was able to package DNA, but was non-infectious. Taken together, thesesuggest that residues R585 and R588 are primarily responsible forheparin sulfate binding and mutation of these residues has little effecton other aspects of the viral life cycle.

Computer modeling using the AAV2 VP3 atomic coordinates revealed thatresidues which contribute to heparin binding form a cluster of fivebasic amino acids on the surface of each three-fold axis of symmetryrelated spike. Three other kinds of mutants were found as well. Mutants,R459A, H509A and H526A/K527A bound heparin as well as wild type but weredefective for transduction. Another mutant, H358A, was defective forcapsid assembly. Finally, a mutant R459A produced significantly lowerlevels of full capsids, suggesting a packaging defect.

Pharmaceutical Compositions

The genetic constructs of the present invention may be prepared in avariety of compositions, and may also be formulated in appropriatepharmaceutical vehicles for administration to human or animal subjects.The AAV molecules of the present invention and compositions comprisingthem provide new and useful therapeutics for the treatment, control, andamelioration of symptoms of a variety of disorders. Moreover,pharmaceutical compositions comprising one or more of the nucleic acidcompounds disclosed herein, provide significant advantages over existingconventional therapies—namely, (1) their reduced side effects, (2) theirincreased efficacy for prolonged periods of time, (3) their ability toincrease patient compliance due to their ability to provide therapeuticeffects following as little as a single administration of the selectedtherapeutic AAV composition to affected individuals. Exemplarypharmaceutical compositions and methods for their administration arediscussed in significant detail hereinbelow.

The invention also provides compositions comprising one or more of thedisclosed vectors, expression systems, virions, viral particles; ormammalian cells. As described hereinbelow, such compositions may furthercomprise a pharmaceutical excipient, buffer, or diluent, and may beformulated for administration to an animal, and particularly a humanbeing. Such compositions may further optionally comprise a liposome, alipid, a lipid complex, a microsphere, a microparticle, a nanosphere, ora nanoparticle, or may be otherwise formulated for administration to thecells, tissues, organs, or body of a mammal in need thereof. Suchcompositions may be formulated for use in therapy, such as for example,in the amelioration, prevention, or treatment of conditions such aspeptide deficiency, polypeptide deficiency, tumor, cancer or othermalignant growth, neurological dysfunction, autoimmune diseases, lupus,cardiovascular disease, pulmonary disease, ischemia, stroke,cerebrovascular accidents, diabetes and diseases of the pancreas, neuraldiseases, including Alzheimer's, Huntington's, Tay-Sach's, andParkinson's diseases, memory loss, trauma, motor impairment, and thelike, as well as biliary, renal or hepatic disease or dysfunction, aswell as musculoskeletal diseases including, for example, arthritis,cystic fibrosis (CF), amyotrophic lateral sclerosis (ALS), multiplesclerosis (MS), muscular dystrophy (MD), and such like, to name only afew.

In certain embodiments, the present invention concerns formulation ofone or more of the rAAV compositions disclosed herein inpharmaceutically acceptable solutions for administration to a cell or ananimal, either alone or in combination with one or more other modalitiesof therapy, and in particular, for therapy of human cells, tissues, anddiseases affecting man.

It will also be understood that, if desired, nucleic acid segments, RNA,DNA or PNA compositions that express one or more of therapeutic geneproducts may be administered in combination with other agents as well,such as, e.g., proteins or polypeptides or variouspharmaceutically-active agents, including one or more systemic ortopical administrations of therapeutic polypeptides, biologically activefragments, or variants thereof. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The rAAV compositions may thus bedelivered along with various other agents as required in the particularinstance. Such compositions may be purified from host cells or otherbiological sources, or alternatively may be chemically synthesized asdescribed herein. Likewise, such compositions may further comprisesubstituted or derivatized RNA, DNA, or PNA compositions.

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the AAVvector-based therapeutic constructs in suitably formulatedpharmaceutical compositions disclosed herein either subcutaneously,intraocularly, intravitreally, parenterally, intravenously,intracerebroventricularly, intramuscularly, intrathecally, orally,intraperitoneally, by oral or nasal inhalation, or by direct injectionto one or more neural cells, nervous tissues, or even by directinjection or administration to the brain, CNS or to the peripheralnervous system. The methods of administration may also include thosemodalities as described in U.S. Pat. No. 5,543,158; U.S. Pat. No.5,641,515 and U.S. Pat. No. 5,399,363 (each of which is specificallyincorporated herein in its entirety by express reference thereto).Solutions of the active compounds as freebase or pharmacologicallyacceptable salts may be prepared in sterile water and may also suitablymixed with one or more surfactants, such as hydroxypropylcellulose.Dispersions may also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms of the AAV-based viral compositions suitablefor injectable use include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions (U.S. Pat. No. 5,466,468, specificallyincorporated herein by reference in its entirety). In all cases the formmust be sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(e.g., glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and/or vegetable oils. Properfluidity may be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialad antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 mL of isotonic NaCl solutionand either added to 1000 mL of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the activeAAV vector-delivered therapeutic polypeptide-encoding DNA fragments inthe required amount in the appropriate solvent with several of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The AAV vector compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human, and in particular, whenadministered to the human eye. The preparation of an aqueous compositionthat contains a protein as an active ingredient is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified.

The amount of AAV compositions and time of administration of suchcompositions will be within the purview of the skilled artisan havingbenefit of the present teachings. It is likely, however, that theadministration of therapeutically-effective amounts of the disclosedcompositions may be achieved by a single administration, such as forexample, a single injection of sufficient numbers of infectiousparticles to provide therapeutic benefit to the patient undergoing suchtreatment. Alternatively, in some circumstances, it may be desirable toprovide multiple, or successive administrations of the AAV vectorcompositions, either over a relatively short, or a relatively prolongedperiod of time, as may be determined by the medical practitioneroverseeing the administration of such compositions. For example, thenumber of infectious particles administered to a mammal may be on theorder of about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or even higher,infectious particles/mL given either as a single dose, or divided intotwo or more administrations as may be required to achieve therapy of theparticular disease or disorder being treated. In fact, in certainembodiments, it may be desirable to administer two or more different AAVvector compositions, either alone, or in combination with one or moreother therapeutic drugs to achieve the desired effects of a particulartherapy regimen.

Liposome-, Nanocapsule-, and Microparticle-Mediated Delivery

In certain embodiments, the inventors contemplate the use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the rAAV vectordelivered gene therapy compositions of the present invention may beformulated for delivery either encapsulated in a lipid particle, aliposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art (see for example, Couvreuret al., 1977; Couvreur, 1988; Lasic, 1998; which describes the use ofliposomes and nanocapsules in the targeted antibiotic therapy forintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; and U.S. Pat.No. 5,741,516, each of which is specifically incorporated herein in itsentirety by express reference thereto). Further, various methods ofliposome and liposome like preparations as potential drug carriers havebeen reviewed (Takakura, 1998; Chandran et al., 1997; Margalit, 1995;U.S. Pat. No. 5,567,434; U.S. Pat. No. 5,552,157; U.S. Pat. No.5,565,213; U.S. Pat. No. 5,738,868 and U.S. Pat. No. 5,795,587, each ofwhich is specifically incorporated herein in its entirety by expressreference thereto).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., 1990; Muller et al., 1990). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes, drugs(Heath and Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989;Fresta and Puglisi, 1996), radiotherapeutic agents (Pikul et al., 1987),enzymes (Imaizumi et al., 1990a; Imaizumi et al., 1990b), viruses(Faller and Baltimore, 1984), transcription factors and allostericeffectors (Nicolau and Gersonde, 1979) into a variety of cultured celllines and animals. In addition, several successful clinical trailsexamining the effectiveness of liposome-mediated drug delivery have beencompleted (Lopez-Berestein et al., 1985a; 1985b; Coune, 1988; Sculier etal., 1988). Furthermore, several studies suggest that the use ofliposomes is not associated with autoimmune responses, toxicity orgonadal localization after systemic delivery (Mori and Fukatsu, 1992).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Liposomes bear resemblance to cellular membranes and are contemplatedfor use in connection with the present invention as carriers for thepeptide compositions. They are widely suitable as both water- andlipid-soluble substances can be entrapped, i.e. in the aqueous spacesand within the bilayer itself, respectively. It is possible that thedrug-bearing liposomes may even be employed for site-specific deliveryof active agents by selectively modifying the liposomal formulation.

In addition to the teachings of Couvreur et al. (1977; 1988), thefollowing information may be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

In addition to temperature, exposure to proteins can alter thepermeability of liposomes. Certain soluble proteins, such as cytochromec, bind, deform and penetrate the bilayer, thereby causing changes inpermeability. Cholesterol inhibits this penetration of proteins,apparently by packing the phospholipids more tightly. It is contemplatedthat the most useful liposome formations for antibiotic and inhibitordelivery will contain cholesterol.

The ability to trap solutes varies between different types of liposomes.For example, MLVs are moderately efficient at trapping solutes, but SUVsare extremely inefficient. SUVs offer the advantage of homogeneity andreproducibility in size distribution, however, and a compromise betweensize and trapping efficiency is offered by large unilamellar vesicles(LUVs). These are prepared by ether evaporation and are three to fourtimes more efficient at solute entrapment than MLVs.

In addition to liposome characteristics, an important determinant inentrapping compounds is the physicochemical properties of the compounditself. Polar compounds are trapped in the aqueous spaces and nonpolarcompounds bind to the lipid bilayer of the vesicle. Polar compounds arereleased through permeation or when the bilayer is broken, but nonpolarcompounds remain affiliated with the bilayer unless it is disrupted bytemperature or exposure to lipoproteins. Both types show maximum effluxrates at the phase transition temperature.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

The fate and disposition of intravenously injected liposomes depend ontheir physical properties, such as size, fluidity, and surface charge.They may persist in tissues for hrs or days, depending on theircomposition, and half lives in the blood range from min to several hrs.Larger liposomes, such as MLVs and LUVs, are taken up rapidly byphagocytic cells of the reticuloendothelial system, but physiology ofthe circulatory system restrains the exit of such large species at mostsites. They can exit only in places where large openings or pores existin the capillary endothelium, such as the sinusoids of the liver orspleen. Thus, these organs are the predominate site of uptake. On theother hand, SUVs show a broader tissue distribution but still aresequestered highly in the liver and spleen. In general, this in vivobehavior limits the potential targeting of liposomes to only thoseorgans and tissues accessible to their large size. These include theblood, liver, spleen, bone marrow, and lymphoid organs.

Targeting is generally not a limitation in terms of the presentinvention. However, should specific targeting be desired, methods areavailable for this to be accomplished. Antibodies may be used to bind tothe liposome surface and to direct the antibody and its drug contents tospecific antigenic receptors located on a particular cell-type surface.Carbohydrate determinants (glycoprotein or glycolipid cell-surfacecomponents that play a role in cell-cell recognition, interaction andadhesion) may also be used as recognition sites as they have potentialin directing liposomes to particular cell types. Mostly, it iscontemplated that intravenous injection of liposomal preparations wouldbe used, but other routes of administration are also conceivable.

Alternatively, the invention provides for pharmaceutically acceptablenanocapsule formulations of the AAV vector-based polynucleotidecompositions of the present invention. Nanocapsules can generally entrapcompounds in a stable and reproducible way (Henry-Michelland et al.,1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoidside effects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 nm) should be designed using polymers ableto be degraded in vivo. Biodegradable polyalkyl-cyanoacrylatenanoparticles that meet these requirements are contemplated for use inthe present invention. Such particles may be are easily made, asdescribed (Couvreur et al., 1980; Couvreur, 1988; zur Muhlen et al.,1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U.S. Pat.No. 5,145,684, each of which is specifically incorporated herein in itsentirety by express reference thereto).

Additional Modes of Delivery

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe disclosed rAAV vector based polynucleotide compositions to a targetcell or animal. Sonophoresis (i.e., ultrasound) has been used anddescribed in U.S. Pat. No. 5,656,016 (specifically incorporated hereinin its entirety by express reference thereto) as a device for enhancingthe rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. No. 5,770,219 and U.S. Pat. No.5,783,208) and feedback-controlled delivery (U.S. Pat. No. 5,697,899),each of which is specifically incorporated herein in its entirety byexpress reference thereto.

Promoters and Enhancers

Recombinant AAV vectors form important aspects of the present invention.The term “expression vector or construct” means any type of geneticconstruct containing a nucleic acid in which part or all of the nucleicacid encoding sequence is capable of being transcribed. In preferredembodiments, expression only includes transcription of the nucleic acid,for example, to generate a biologically-active therapeutic peptides,polypeptides, proteins, antisense molecules, or catalytic RNA ribozymesfrom a transcribed gene.

Particularly useful vectors are contemplated to be those vectors inwhich the nucleic acid segment to be transcribed is positioned under thetranscriptional control of a promoter. A “promoter” refers to a DNAsequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrases “operatively positioned,” “undercontrol” or “under transcriptional control” means that the promoter isin the correct location and orientation in relation to the nucleic acidto control RNA polymerase initiation and expression of the gene.

In preferred embodiments, it is contemplated that certain advantageswill be gained by positioning the coding DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with an cytokine or serpin-encoding genein its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or mammalian cell.

Naturally, it will be important to employ a promoter that effectivelydirects the expression of the serpin or cytokine-encoding DNA segment inthe cell type, organism, or even animal, chosen for expression. The useof promoter and cell type combinations for protein expression isgenerally known to those of skill in the art of molecular biology, forexample, see Sambrook et al. (1989), incorporated herein by reference.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high-level expression ofthe introduced DNA segment, or the promoters may direct tissue- orcell-specific expression of the therapeutic constructs, such as, forexample, an islet cell- or pancreas-specific promoter such as theinsulin promoter.

At least one module in a promoter functions to position the start sitefor RNA synthesis. The best-known example of this is the TATA box, butin some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 byupstream of the start site, although a number of promoters have beenshown to contain functional elements downstream of the start site aswell. The spacing between promoter elements frequently is flexible, sothat promoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 by apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the serpin or cytokine-polypeptide encoding nucleic acidsegment in the targeted cell. Thus, where a human cell is targeted, itis preferable to position the nucleic acid coding region adjacent to andunder the control of a promoter that is capable of being expressed in ahuman cell. Generally speaking, such a promoter might include either ahuman or viral promoter, such as a CMV or an HSV promoter. In certainaspects of the invention, β-actin, and in particular, chicken β-actinpromoters have been shown to be particularly preferred for certainembodiments of the invention.

In various other embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters that are well known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose. A variety of promoterelements have been described in Tables 1 and 2 that may be employed, inthe context of the present invention, to regulate the expression of thepresent serpin or cytokine-encoding nucleic acid segments comprisedwithin the recombinant AAV vectors of the present invention.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression. Use ofa T3, T7 or SP6 cytoplasmic expression system is another possibleembodiment. Eukaryotic cells can support cytoplasmic transcription fromcertain bacterial promoters if the appropriate bacterial polymerase isprovided, either as part of the delivery complex or as an additionalgenetic expression construct.

TABLE 1 ILLUSTRATIVE PROMOTER AND ENHANCER ELEMENTS PROMOTER/ENHANCERREFERENCE Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles etal., 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986,1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al.,1988; Porton et al.; 1990 Immunoglobulin Light Chain Queen andBaltimore, 1983; Picard and Schaffner, 1984 T-Cell Receptor Luria etal., 1987; Winoto and Baltimore, 1989; Redondo et al.; 1990 HLA DQ a andDQ β Sullivan and Peterlin, 1987 β-Interferon Goodbourn et al., 1986;Fujita et al., 1987; Goodbourn and Maniatis, 1988 Interleukin-2 Greeneet al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al.,1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-Dra Sherman etal., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 Muscle CreatineKinase Jaynes et al., 1988; Horlick and Benfield, 1989; Johnson et al.,1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Omitz etal., 1987 Metallothionein Karin et al., 1987; Culotta and Hamer, 1989Collagenase Pinkert et al., 1987; Angel et al., 1987 Albumin GenePinkert et al., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godboutet al., 1988; Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987;Perez-Stable and Constantini, 1990 β-Globin Trudel and Constantini, 1987e-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985Insulin Edlund et al., 1985 Neural Cell Adhesion Molecule Hirsh et al.,1990 (NCAM) α_(1-Antitrypain) Latimer et al., 1990 H2B (TH2B) HistoneHwang et al., 1990 Mouse or Type I Collagen Ripe et al., 1989Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) RatGrowth Hormone Larsen et al., 1986 Human Serum Amyloid A (SAA) Edbrookeet al., 1989 Troponin I (TN I) Yutzey et al., 1989 Platelet-DerivedGrowth Factor Pech et al., 1989 Duchenne Muscular Dystrophy Klamut etal., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh andLockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbraand Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek etal., 1987; Kuhl et al., 1987; Schaffner et al., 1988 PolyomaSwartzendruber and Lehman, 1975; Vasseur et al., 1980; Katinka et al.,1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers etal., 1984; Hen et al., 1986; Satake et al., 1988; Campbell andVillarreal, 1988 Retroviruses Kriegler and Botchan, 1982, 1983; Levinsonet al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander and Haseltine, 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman and Rotter, 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos andWilkie, 1983; Spalholz et al., 1985; Lusky and Botchan, 1986; Cripe etal., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens andHentschel, 1987 Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel andSiddiqui, 1986; Shaul and Ben-Levy, 1987; Spandau and Lee, 1988; Vanniceand Levinson, 1988 Human Immunodeficiency Virus Muesing et al., 1987;Hauber and Cullan, 1988; Jakobovits et al., 1988; Feng and Holland,1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989;Laspia et al., 1989; Sharp and Marciniak, 1989; Braddock et al., 1989Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foecking andHofstetter, 1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinnet al., 1989

TABLE 2 INDUCIBLE ELEMENTS ELEMENT INDUCER REFERENCES MT II PhorbolEster (TFA) Palmiter et al., 1982; Heavy metals Haslinger and Karin,1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987,Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV(mouse mammary tumor virus) Glucocorticoids Huang et al., 1981; Lee etal., 1981; Majors and Varmus, 1983; Chandler et al., 1983; Lee et al.,1984; Ponta et al., 1985; Sakai et al., 1988 β-Interferon poly(rI)xTavernier et al., 1983 poly(rc) Adenovirus 5 E2 Ela Imperiale andNevins, 1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987aStromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester(TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle DiseaseVirus GRP78 Gene A23187 Resendez et al., 1988 α-2-Macroglobulin IL-6Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I GeneH-2κb Interferon Blanar et al., 1989 HSP70 Ela, SV40 Large T AntigenTaylor et al., 1989; Taylor and Kingston, 1990a, b Proliferin PhorbolEster-TPA Mordacq and Linzer, 1989 Tumor Necrosis Factor FMA Hensel etal., 1989 Thyroid Stimulating Hormone α Gene Thyroid Hormone Chatterjeeet al., 1989

As used herein, the terms “engineered” and “recombinant” cells areintended to refer to a cell into which an exogenous DNA segment, such asDNA segment that leads to the transcription of a biologically-activeserpin or cytokine polypeptide or a ribozyme specific for such abiologically-active serpin or cytokine polypeptide product, has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells, which do not contain a recombinantlyintroduced exogenous DNA segment. Engineered cells are thus cells havingDNA segment introduced through the hand of man.

To express a biologically-active serpin or cytokine encoding gene inaccordance with the present invention one would prepare an rAAVexpression vector that comprises a biologically-active serpin orcytokine polypeptide-encoding nucleic acid segment under the control ofone or more promoters. To bring a sequence “under the control of” apromoter, one positions the 5′-end of the transcription initiation siteof the transcriptional reading frame generally between about 1 and about50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. The“upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded polypeptide. This is the meaning of“recombinant expression” in this context. Particularly preferredrecombinant vector constructs are those that comprise an rAAV vector.Such vectors are described in detail herein.

Mutagenesis and Preparation of Modified Nucleotide Compositions

In certain embodiments, it may be desirable to prepared modifiednucleotide compositions, such as, for example, in the generation of thenucleic acid segments that encode either parts of the AAV vector itself,or the promoter, or even the therapeutic gene delivered by such rAAVvectors. Various means exist in the art, and are routinely employed bythe artisan to generate modified nucleotide compositions.

Site-specific mutagenesis is a technique useful in the preparation andtesting of sequence variants by introducing one or more nucleotidesequence changes into the DNA. Site-specific mutagenesis allows theproduction of mutants through the use of specific oligonucleotidesequences which encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed.Typically, a primer of about 17 to 25 nucleotides in length ispreferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. As will be appreciated, the technique typically employs abacteriophage vector that exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage. These phage vectors arecommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis, which eliminates the step of transferringthe gene of interest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector that includes within its sequence a DNA sequence encoding thedesired ribozyme or other nucleic acid construct. An oligonucleotideprimer bearing the desired mutated sequence is synthetically prepared.This primer is then annealed with the single-stranded DNA preparation,and subjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected that include recombinant vectors bearing the mutated sequencearrangement.

The preparation of sequence variants of the selected nucleic acidsequences using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting, asthere are other ways in which sequence variants may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

Nucleic Acid Amplification

In certain embodiments, it may be necessary to employ one or morenucleic acid amplification techniques to produce the nucleic acidsegments of the present invention. Various methods are well-known toartisans in the field, including for example, those techniques describedherein:

Nucleic acid, used as a template for amplification, may be isolated fromcells contained in the biological sample according to standardmethodologies (Sambrook et al., 1989). The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a complementary DNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to the ribozymes or conserved flanking regions arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer”, as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with oneor more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (e.g., Affymax technology).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of thebest-known amplification methods is the polymerase chain reaction(referred to as PCR™), which is described in detail in U.S. Pat. No.4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159 (each ofwhich is specifically incorporated herein in its entirety by expressreference thereto).

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described (Sambrook etal., 1989). Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin Int. Pat. Appl. Publ. No. WO 90/07641 (specifically incorporatedherein in its entirety by express reference thereto). Polymerase chainreaction methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EP Appl. No. 320308 (specifically incorporated herein inits entirety by express reference thereto). In LCR, two complementaryprobe pairs are prepared, and in the presence of the target sequence,each pair will bind to opposite complementary strands of the target suchthat they abut. In the presence of a ligase, the two probe pairs willlink to form a single unit. By temperature cycling, as in PCR™, boundligated units dissociate from the target and then serve as “targetsequences” for ligation of excess probe pairs. U.S. Pat. No. 4,883,750(specifically incorporated herein in its entirety by express referencethereto) describes a method similar to LCR for binding probe pairs to atarget sequence.

Qβ Replicase (QβR), described in Int. Pat. Appl. No. PCT/US87/00880(specifically incorporated herein in its entirety by express referencethereto), may also be used as still another amplification method in thepresent invention. In this method, a replicative sequence of RNA thathas a region complementary to that of a target is added to a sample inthe presence of an RNA polymerase. The polymerase will copy thereplicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA), described in U.S. Pat. Nos.5,455,166, 5,648,211, 5,712,124 and 5,744,311 (each of which isspecifically incorporated herein in its entirety by express referencethereto), is another method of carrying out isothermal amplification ofnucleic acids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation. A similar method, called Repair ChainReaction (RCR), involves annealing several probes throughout a regiontargeted for amplification, followed by a repair reaction in which onlytwo of the four bases are present. The other two bases can be added asbiotinylated derivatives for easy detection. A similar approach is usedin SDA. Target specific sequences can also be detected using a cyclicprobe reaction (CPR). In CPR, a probe having 3′ and 5′ sequences ofnon-specific DNA, and a middle sequence of specific RNA is hybridized toDNA that is present in a sample. Upon hybridization, the reaction istreated with RNase H, and the products of the probe identified asdistinctive products that are released after digestion. The originaltemplate is annealed to another cycling probe and the reaction isrepeated.

Still another amplification methods described in GB Application No. 2202 328, and in Int. Pat. Appl. No. PCT/US89/01025 (each of which isspecifically incorporated herein in its entirety by express referencethereto), may be used in accordance with the present invention. In theformer application, “modified” primers are used in a PCR™-like,template- and enzyme-dependent synthesis. The primers may be modified bylabeling with a capture moiety (e.g., biotin) and/or a detector moiety(e.g., enzyme). In the latter application, an excess of labeled probesis added to a sample. In the presence of the target sequence, the probebinds and is cleaved catalytically. After cleavage, the target sequenceis released intact to be bound by excess probe. Cleavage of the labeledprobe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR Gingeras et al., PCT Intl. Pat. Appl.Publ. No. WO 88/10315 (specifically incorporated herein in its entiretyby express reference thereto). In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer that has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by an RNA polymerase such as T7 or SP6. Inan isothermal cyclic reaction, the RNAs are reverse transcribed intosingle stranded DNA, which is then converted to double-stranded DNA, andthen transcribed once again with an RNA polymerase such as T7 or SP6.The resulting products, whether truncated or complete, indicate targetspecific sequences.

Davey et al., EPA No. 329 822 (specifically incorporated herein in itsentirety by express reference thereto) disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be usedin accordance with the present invention. The ssRNA is a template for afirst primer oligonucleotide, which is elongated by reversetranscriptase (RNA-dependent DNA polymerase). The RNA is then removedfrom the resulting DNA:RNA duplex by the action of ribonuclease H(RNaseH, an RNase specific for RNA in duplex with either DNA or RNA). Theresultant ssDNA is a template for a second primer, which also includesthe sequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., Int. Pat. Appl. Publ. No. WO 89/06700 (specificallyincorporated herein in its entirety by express reference thereto)disclose a nucleic acid sequence amplification scheme based on thehybridization of a promoter/primer sequence to a target single-strandedDNA (“ssDNA”) followed by transcription of many RNA copies of thesequence. This scheme is not cyclic, i.e., new templates are notproduced from the resultant RNA transcripts. Other amplification methodsinclude “RACE” and “one-sided PCR™” (Frohman, 1990; specificallyincorporated herein in its entirety by express reference thereto).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (see e.g., Sambrook et al., 1989).

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols. See Sambrook et al., 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and non-covalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721(specifically incorporated herein in its entirety by express referencethereto), which discloses an apparatus and method for the automatedelectrophoresis and transfer of nucleic acids. The apparatus permitselectrophoresis and blotting without external manipulation of the geland is ideally suited to carrying out methods according to the presentinvention.

Methods of Nucleic Acid Delivery and DNA Transfection

In certain embodiments, it is contemplated that one or more RNA, DNA,PNAs and/or substituted polynucleotide compositions disclosed hereinwill be used to transfect an appropriate host cell. Technology forintroduction of PNAs, RNAs, and DNAs into cells is well known to thoseof skill in the art.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Wong and Neumann, 1982; Fromm et al., 1985;Tur-Kaspa et al., 1986; Potter et al., 1984; Suzuki et al., 1998;Vanbever et al., 1998), direct microinjection (Capecchi, 1980; Harlandand Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;Fraley et al., 1979; Takakura, 1998) and lipofectamine-DNA complexes,cell sonication (Fechheimer et al., 1987), gene bombardment using highvelocity microprojectiles (Yang et al., 1990; Klein et al., 1992), andreceptor-mediated transfection (Curiel et al., 1991; Wagner et al.,1992; Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

Expression Vectors

The present invention contemplates a variety of AAV-based expressionsystems, and vectors. In one embodiment the preferred AAV expressionvectors comprise at least a first nucleic acid segment that encodes atherapeutic peptide, protein, or polypeptide. In another embodiment, thepreferred AAV expression vectors disclosed herein comprise at least afirst nucleic acid segment that encodes an antisense molecule. Inanother embodiment, a promoter is operatively linked to a sequenceregion that encodes a functional mRNA, a tRNA, a ribozyme or anantisense RNA.

As used herein, the term “operatively linked” means that a promoter isconnected to a functional RNA in such a way that the transcription ofthat functional RNA is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a functional RNA are well known inthe art.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depend directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the functional RNA to which itis operatively linked.

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

Biological Functional Equivalents

Modification and changes to the structure of the polynucleotides andpolypeptides of wild-type rAAV vectors to provide the improved rAAVvirions as described in the present invention to obtain functional viralvectors that possess desirable characteristics, particularly withrespect to improved delivery of therapeutic gene constructs to selectedmammalian cell, tissues, and organs for the treatment, prevention, andprophylaxis of various diseases and disorders, as well as means for theamelioration of symptoms of such diseases, and to facilitate theexpression of exogenous therapeutic and/or prophylactic polypeptides ofinterest via rAAV vector-mediated gene therapy. As mentioned above, oneof the key aspects of the present invention is the creation of one ormore mutations into specific polynucleotide sequences that encode one ormore of the therapeutic agents encoded by the disclosed rAAV constructs.In certain circumstances, the resulting polypeptide sequence is alteredby these mutations, or in other cases, the sequence of the polypeptideis unchanged by one or more mutations in the encoding polynucleotide toproduce modified vectors with improved properties for effecting genetherapy in mammalian systems.

When it is desirable to alter the amino acid sequence of a polypeptideto create an equivalent, or even an improved, second-generationmolecule, the amino acid changes may be achieved by changing one or moreof the codons of the encoding DNA sequence, according to Table 3.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the polynucleotide sequences disclosed herein,without appreciable loss of their biological utility or activity.

TABLE 3 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics(Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can bemade effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein in its entirety by express referencethereto), states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within ±1 are particularlypreferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take several of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Therapeutic and Diagnostic Kits

The invention also encompasses one or more of the modified rAAV vectorcompositions described herein together with one or morepharmaceutically-acceptable excipients, carriers, diluents, adjuvants,and/or other components, as may be employed in the formulation ofparticular rAAV-polynucleotide delivery formulations, and in thepreparation of therapeutic agents for administration to a mammal, and inparticularly, to a human. In particular, such kits may comprise one ormore of the disclosed rAAV compositions in combination with instructionsfor using the viral vector in the treatment of such disorders in amammal, and may typically further include containers prepared forconvenient commercial packaging.

As such, preferred animals for administration of the pharmaceuticalcompositions disclosed herein include mammals, and particularly humans.Other preferred animals include murines, bovines, equines, porcines,canines, and felines. The composition may include partially orsignificantly purified rAAV compositions, either alone, or incombination with one or more additional active ingredients, which may beobtained from natural or recombinant sources, or which may be obtainablenaturally or either chemically synthesized, or alternatively produced invitro from recombinant host cells expressing DNA segments encoding suchadditional active ingredients.

Therapeutic kits may also be prepared that comprise at least one of thecompositions disclosed herein and instructions for using the compositionas a therapeutic agent. The container means for such kits may typicallycomprise at least one vial, test tube, flask, bottle, syringe or othercontainer means, into which the disclosed rAAV composition(s) may beplaced, and preferably suitably aliquoted. Where a second therapeuticpolypeptide composition is also provided, the kit may also contain asecond distinct container means into which this second composition maybe placed. Alternatively, the plurality of therapeutic biologicallyactive compositions may be prepared in a single pharmaceuticalcomposition, and may be packaged in a single container means, such as avial, flask, syringe, bottle, or other suitable single container means.The kits of the present invention will also typically include a meansfor containing the vial(s) in close confinement for commercial sale,such as, e.g., injection or blow-molded plastic containers into whichthe desired vial(s) are retained.

Ribozymes

As mentioned above, one aspect of the invention concerns the use of themodified capsid vectors to deliver catalytic RNA molecules (ribozymes)to selected mammalian cells and tissues to effect a reduction orelimination of expression of one or more native DNA or mRNA molecules,so as to prevent or reduce the amount of the translation product of suchmRNAs. Ribozymes are biological catalysts consisting of only RNA. Theypromote a variety of reactions involving RNA and DNA molecules includingsite-specific cleavage, ligation, polymerization, and phosphorylexchange (Cech, 1989; Cech, 1990). Ribozymes fall into three broadclasses: (1) RNAse P, (2) self-splicing introns, and (3) self-cleavingviral agents. Self-cleaving agents include hepatitis delta virus andcomponents of plant virus satellite RNAs that sever the RNA genome aspart of a rolling-circle mode of replication. Because of their smallsize and great specificity, ribozymes have the greatest potential forbiotechnical applications. The ability of ribozymes to cleave other RNAmolecules at specific sites in a catalytic manner has brought them intoconsideration as inhibitors of viral replication or of cellproliferation and gives them potential advantage over antisense RNA.Indeed, ribozymes have already been used to cleave viral targets andoncogene products in living cells (Koizumi et al., 1992; Kashani-Sabetet al., 1992; Taylor and Rossi, 1991; von-Weizsacker et al., 1992;Ojwang et al., 1992; Stephenson and Gibson, 1991; Yu et al., 1993; Xingand Whitton, 1993; Yu et al., 1995; Little and Lee, 1995).

Two kinds of ribozymes have been employed widely, hairpins andhammerheads. Both catalyze sequence-specific cleavage resulting inproducts with a 5N hydroxyl and a 2N,3N-cyclic phosphate. Hammerheadribozymes have been used more commonly, because they impose fewrestrictions on the target site. Hairpin ribozymes are more stable and,consequently, function better than hammerheads at physiologictemperature and magnesium concentrations.

A number of patents have issued describing various ribozymes and methodsfor designing ribozymes. See, for example, U.S. Pat. Nos. 5,646,031;5,646,020; 5,639,655; 5,093,246; 4,987,071; 5,116,742; and 5,037,746(each of which is specifically incorporated herein in its entirety byexpress reference thereto). However, the ability of ribozymes to providetherapeutic benefit in vivo has not yet been demonstrated.

Although proteins traditionally have been used for catalysis of nucleicacids, another class of macromolecules has emerged as useful in thisendeavor. Ribozymes are RNA-protein complexes that cleave nucleic acidsin a site-specific fashion. Ribozymes have specific catalytic domainsthat possess endonuclease activity (Kim and Cech, 1987; Gerlach et al.,1987; Forster and Symons, 1987). For example, a large number ofribozymes accelerate phosphoester transfer reactions with a high degreeof specificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855(specifically incorporated herein in its entirety by express referencethereto) reports that certain ribozymes can act as endonucleases with asequence-specificity greater than that of known ribonucleases andapproaching that of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.,1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied; the altered genesincluded the oncogenes H-ras, c-fos and genes of HIV. Most of this workinvolved the modification of a target mRNA, based on a specific mutantcodon that is cleaved by a specific ribozyme.

Six basic varieties of naturally occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans- (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofan enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., 1992). Thus, thespecificity of action of a ribozyme is greater than that of an antisenseoligonucleotide binding the same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992).Examples of hairpin motifs are described by Hampel et al. (Eur. Pat.Appl. Publ. No. EP 0360257), Hampel and Tritz (1989), Hampel et al.(1990) and U.S. Pat. No. 5,631,359 (specifically incorporated herein inits entirety by express reference thereto). An example of the hepatitisδ virus motif is described by Perrotta and Been (1992); an example ofthe RNaseP motif is described by Guerrier-Takada et al. (1983);Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, 1990; Saville and Collins, 1991; Collins and Olive, 1993); andan example of the Group I intron is described in U.S. Pat. No. 4,987,071(specifically incorporated herein in its entirety by express referencethereto). All that is important in an enzymatic nucleic acid molecule ofthis invention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule. Thus theribozyme constructs need not be limited to specific motifs mentionedherein.

In certain embodiments, it may be important to produce enzymaticcleaving agents that exhibit a high degree of specificity for the RNA ofa desired target, such as one of the sequences disclosed herein. Theenzymatic nucleic acid molecule is preferably targeted to a highlyconserved sequence region of a target mRNA. Such enzymatic nucleic acidmolecules can be delivered exogenously to specific cells as required,although in preferred embodiments the ribozymes are expressed from DNAor RNA vectors that are delivered to specific cells.

Small enzymatic nucleic acid motifs (e.g., of the hammerhead or thehairpin structure) may also be used for exogenous delivery. The simplestructure of these molecules increases the ability of the enzymaticnucleic acid to invade targeted regions of the mRNA structure.Alternatively, catalytic RNA molecules can be expressed within cellsfrom eukaryotic promoters (e.g., Scanlon et al., 1991; Kashani-Sabet etal., 1992; Dropulic et al., 1992; Weerasinghe et al., 1991; Ojwang etal., 1992; Chen et al., 1992; Sarver et al., 1990). Those skilled in theart realize that any ribozyme can be expressed in eukaryotic cells fromthe appropriate DNA vector. The activity of such ribozymes can beaugmented by their release from the primary transcript by a secondribozyme (PCT Intl. Pat. Appl. Publ. No. WO 93/23569, and PCT Intl. Pat.Appl. Publ. No. WO 94/02595, each of which is specifically incorporatedherein in its entirety by express reference thereto; Ohkawa et al.,1992; Taira et al., 1991; and Ventura et al., 1993).

Ribozymes may be added directly, or can be complexed with cationiclipids, lipid complexes, packaged within liposomes, or otherwisedelivered to target cells. The RNA or RNA complexes can be locallyadministered to relevant tissues ex vivo, or in vivo through injection,aerosol inhalation, infusion pump or stent, with or without theirincorporation in biopolymers.

Ribozymes may be designed as described in PCT Intl. Pat. Appl. Publ. No.WO 93/23569 and PCT Intl. Pat. Appl. Publ. No. WO 94/02595 (each ofwhich is specifically incorporated herein in its entirety by expressreference thereto) and synthesized to be tested in vitro and in vivo, asdescribed. Such ribozymes can also be optimized for delivery. Whilespecific examples are provided, those in the art will recognize thatequivalent RNA targets in other species can be utilized when necessary.

Hammerhead or hairpin ribozymes may be individually analyzed by computerfolding (Jaeger et al., 1989) to assess whether the ribozyme sequencesfold into the appropriate secondary structure, as described herein.Those ribozymes with unfavorable intramolecular interactions between thebinding arms and the catalytic core are eliminated from consideration.Varying binding arm lengths can be chosen to optimize activity.Generally, at least 5 or so bases on each arm are able to bind to, orotherwise interact with, the target RNA.

Ribozymes of the hammerhead or hairpin motif may be designed to annealto various sites in the mRNA message, and can be chemically synthesized.The method of synthesis used follows the procedure for normal RNAsynthesis as described in Usman et al. (1987) and in Scaringe et al.(1990) and makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end, and phosphoramidites atthe 3′-end. Average stepwise coupling yields are typically >98%. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct anactive ribozyme (Chowrira and Burke, 1992). Ribozymes may be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-o-methyl, 2′-H(for a review see e.g., Usman and Cedergren, 1992). Ribozymes may bepurified by gel electrophoresis using general methods or byhigh-pressure liquid chromatography and resuspended in water.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990;Pieken et al., 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ.No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688 (each of which is specifically incorporated herein inits entirety by express reference thereto), which describe variouschemical modifications that can be made to the sugar moieties ofenzymatic RNA molecules), modifications which enhance their efficacy incells, and removal of stem II bases to shorten RNA synthesis times andreduce chemical requirements.

A preferred means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used, providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gaoand Huang, 1993; Lieber et al., 1993; Zhou et al., 1990). Ribozymesexpressed from such promoters can function in mammalian cells(Kashani-Sabet et al., 1992; Ojwang et al., 1992; Chen et al., 1992; Yuet al., 1993; L'Huillier et al., 1992; Lisziewicz et al., 1993).Although incorporation of the present ribozyme constructs intoadeno-associated viral vectors is preferred, such transcription unitscan be incorporated into a variety of vectors for introduction intomammalian cells, including but not restricted to, plasmid DNA vectors,other viral DNA vectors (such as adenovirus vectors, etc.), or viral RNAvectors (such as retroviral, semliki forest virus, sindbis virusvectors, etc.).

Sullivan et al. (PCT Intl. Pat. Appl. Publ. No. WO 94/02595;specifically incorporated herein in its entirety by express referencethereto) describes general methods for delivery of enzymatic RNAmolecules. Ribozymes may be administered to cells by a variety ofmethods known to those familiar to the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres. For someindications, ribozymes may be directly delivered ex vivo to cells ortissues with or without the aforementioned vehicles. Alternatively, theRNA/vehicle combination may be locally delivered by direct inhalation,by direct injection or by use of a catheter, infusion pump or stent.Other routes of delivery include, but are not limited to, intravascular,intramuscular, subcutaneous or joint injection, aerosol inhalation, oral(tablet or pill form), topical, systemic, ocular, intraocular, retinal,subretinal, intraperitoneal, intracerebroventricular, intrathecaldelivery, and/or direct injection to one or more tissues of the brain.More detailed descriptions of ribozyme and rAAV vector delivery andadministration are provided in PCT Intl. Pat. Appl. Publ. No. WO94/02595 and PCT Intl. Pat. Appl. Publ. No. WO 93/23569, each of whichis specifically incorporated herein in its entirety by express referencethereto.

Ribozymes and the AAV vectored-constructs of the present invention maybe used to inhibit gene expression and define the role (essentially) ofspecified gene products in the progression of one or more neuraldiseases, dysfunctions, cancers, and/or disorders. In this manner, othergenetic targets may be defined as important mediators of the disease.These studies lead to better treatment of the disease progression byaffording the possibility of combination therapies (e.g., multipleribozymes targeted to different genes, ribozymes coupled with knownsmall molecule inhibitors, or intermittent treatment with combinationsof ribozymes and/or other chemical or biological molecules).

Antisense Oligonucleotides

In certain embodiments, the AAV constructs of the invention will findutility in the delivery of antisense oligonucleotides andpolynucleotides for inhibiting the expression of a selected mammalianmRNA in neural cells.

In the art the letters, A, G, C, T, and U respectively indicatenucleotides in which the nucleoside is Adenosine (Ade), Guanosine (Gua),Cytidine (Cyt), Thymidine (Thy), and Uridine (Ura). As used in thespecification and claims, compounds that are “antisense” to a particularPNA, DNA or mRNA “sense” strand are nucleotide compounds that have anucleoside sequence that is complementary to the sense strand. It willbe understood by those skilled in the art that the present inventionbroadly includes oligonucleotide compounds that are capable of bindingto the selected DNA or mRNA sense strand. It will also be understoodthat mRNA includes not only the ribonucleotide sequences encoding aprotein, but also regions including the 5′-untranslated region, the3′-untranslated region, the 5′-cap region and the intron/exon junctionregions.

The invention includes compounds which are not strictly antisense; thecompounds of the invention also include those oligonucleotides that mayhave some bases that are not complementary to bases in the sense strandprovided such compounds have sufficient binding affinity for theparticular DNA or mRNA for which an inhibition of expression is desired.In addition, base modifications or the use of universal bases such asinosine in the oligonucleotides of the invention are contemplated withinthe scope of the subject invention.

The antisense compounds may have some or all of the phosphates in thenucleotides replaced by phosphorothioates (X═S) or methylphosphonates(X═CH₃) or other C₁₋₄ alkylphosphonates. The antisense compoundsoptionally may be further differentiated from native DNA by replacingone or both of the free hydroxy groups of the antisense molecule withC₁₋₄ alkoxy groups (R═C₁₋₄ alkoxy). As used herein, C₁₋₄ alkyl means abranched or unbranched hydrocarbon having 1 to 4 carbon-atoms.

The disclosed antisense compounds also may be substituted at the 3′and/or 5′ ends by a substituted acridine derivative. As used herein,“substituted acridine,” means any acridine derivative capable ofintercalating nucleotide strands such as DNA. Preferred substitutedacridines are 2-methoxy-6-chloro-9-pentylaminoacridine,N-(6-chloro-2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-3-aminopropanol,andN-(6-chloro2-methoxyacridinyl)-O-methoxydiisopropylaminophosphinyl-5-aminopentanol.Other suitable acridine derivatives are readily apparent to personsskilled in the art. Additionally, as used herein “P(O)(O)-substitutedacridine” means a phosphate covalently linked to a substitute acridine.

As used herein, the term “nucleotides” includes nucleotides in which thephosphate moiety is replaced by phosphorothioate or alkylphosphonate andthe nucleotides may be substituted by substituted acridines.

In one embodiment, the antisense compounds of the invention differ fromnative DNA by the modification of the phosphodiester backbone to extendthe life of the antisense molecule. For example, the phosphates can bereplaced by phosphorothioates. The ends of the molecule may also beoptimally substituted by an acridine derivative that intercalatesnucleotide strands of DNA. PCT Intl. Pat. Appl. Publ. No. WO 98/13526and U.S. Pat. No. 5,849,902 (each of which is specifically incorporatedherein in its entirety by express reference thereto) describe a methodof preparing three component chimeric antisense compositions, anddiscuss many of the currently available methodologies for synthesis ofsubstituted oligonucleotides having improved antisense characteristicsand/or half-life.

The reaction scheme involves ¹H-tetrazole-catalyzed coupling ofphosphoramidites to give phosphate intermediates that are subsequentlyreacted with sulfur in 2,6-lutidine to generate phosphate compounds.Oligonucleotide compounds are prepared by treating the phosphatecompounds with thiophenoxide (1:2:2thiophenol/triethylamine/tetrahydrofuran, room temperature, 1 hr). Thereaction sequence is repeated until an oligonucleotide compound of thedesired length has been prepared. The compounds are cleaved from thesupport by treating with ammonium hydroxide at room temperature for 1 hrand then are further deprotected by heating at about 50° C. overnight toyield preferred antisense compounds.

Selection of antisense compositions specific for a given gene sequenceis based upon analysis of the chosen target sequence and determinationof secondary structure, T_(m), binding energy, relative stability, andantisense compositions were selected based upon their relative inabilityto form dimers, hairpins, or other secondary structures that wouldreduce or prohibit specific binding to the target mRNA in a host cell.Highly preferred target regions of the mRNA, are those that are at ornear the AUG translation initiation codon, and those sequences that weresubstantially complementary to 5′ regions of the mRNA. These secondarystructure analyses and target site selection considerations wereperformed using v.4 of the OLIGO primer analysis software (Rychlik,1997) and the BLASTN 2.0.5 algorithm software (Altschul et al., 1997).

Exemplary Definitions

In accordance with the present invention, polynucleotides, nucleic acidsegments, nucleic acid sequences, and the like, include, but are notlimited to, DNAs (including and not limited to genomic or extragenomicDNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but notlimited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleicacid segments either obtained from native sources, chemicallysynthesized, modified, or otherwise prepared in whole or in part by thehand of man.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andcompositions similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and compositions are described herein. For purposes of thepresent invention, the following terms are defined below:

A, an: In accordance with long standing patent law convention, the words“a” and “an” when used in this application, including the claims,denotes “one or more”.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a polynucleotide such as astructural gene to synthesize the encoded peptide or polypeptide.

Promoter: a term used to generally describe the region or regions of anucleic acid sequence that regulates transcription.

Regulatory element: a term used to generally describe the region orregions of a nucleic acid sequence that regulates transcription.

Structural gene: A gene or sequence region that is expressed to producean encoded peptide or polypeptide.

Transformation: A process of introducing an exogenous polynucleotidesequence (e.g., a vector, a recombinant DNA or RNA molecule) into a hostcell or protoplast in which that exogenous nucleic acid segment isincorporated into at least a first chromosome or is capable ofautonomous replication within the transformed host cell. Transfection,electroporation, and naked nucleic acid uptake all represent examples oftechniques used to transform a host cell with one or morepolynucleotides.

Transformed cell: A host cell whose nucleic acid complement has beenaltered by the introduction of one or more exogenous polynucleotidesinto that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell, or from the progeny or offspring ofany generation of such a transformed host cell.

Vector: A nucleic acid molecule (typically comprised of DNA) capable ofreplication in a host cell and/or to which another nucleic acid segmentcan be operatively linked so as to bring about replication of theattached segment. A plasmid, cosmid, or a virus is an exemplary vector.

The terms “substantially corresponds to”, “substantially homologous”, or“substantial identity” as used herein denotes a characteristic of anucleic acid or an amino acid sequence, wherein a selected nucleic acidor amino acid sequence has at least about 70 or about 75 percentsequence identity as compared to a selected reference nucleic acid oramino acid sequence. More typically, the selected sequence and thereference sequence will have at least about 76, 77, 78, 79, 80, 81, 82,83, 84 or even 85 percent sequence identity, and more preferably atleast about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequenceidentity. More preferably still, highly homologous sequences often sharegreater than at least about 96, 97, 98, or 99 percent sequence identitybetween the selected sequence and the reference sequence to which it wascompared. The percentage of sequence identity may be calculated over theentire length of the sequences to be compared, or may be calculated byexcluding small deletions or additions which total less than about 25percent or so of the chosen reference sequence. The reference sequencemay be a subset of a larger sequence, such as a portion of a gene orflanking sequence, or a repetitive portion of a chromosome. However, inthe case of sequence homology of two or more polynucleotide sequences,the reference sequence will typically comprise at least about 18-25nucleotides, more typically at least about 26 to 35 nucleotides, andeven more typically at least about 40, 50, 60, 70, 80, 90, or even 100or so nucleotides. Desirably, which highly homologous fragments aredesired, the extent of percent identity between the two sequences willbe at least about 80%, preferably at least about 85%, and morepreferably about 90% or 95% or higher, as readily determined by one ormore of the sequence comparison algorithms well-known to those of skillin the art, such as e.g., the FASTA program analysis described byPearson and Lipman (1988).

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by the hand of man in alaboratory is naturally-occurring. As used herein, laboratory strains ofrodents that may have been selectively bred according to classicalgenetics are considered naturally occurring animals.

As used herein, a “heterologous” is defined in relation to apredetermined referenced gene sequence. For example, with respect to astructural gene sequence, a heterologous promoter is defined as apromoter which does not naturally occur adjacent to the referencedstructural gene, but which is positioned by laboratory manipulation.Likewise, a heterologous gene or nucleic acid segment is defined as agene or segment that does not naturally occur adjacent to the referencedpromoter and/or enhancer elements.

“Transcriptional regulatory element” refers to a polynucleotide sequencethat activates transcription alone or in combination with one or moreother nucleic acid sequences. A transcriptional regulatory element can,for example, comprise one or more promoters, one or more responseelements, one or more negative regulatory elements, and/or one or moreenhancers.

As used herein, a “transcription factor recognition site” and a“transcription factor binding site” refer to a polynucleotidesequence(s) or sequence motif(s) which are identified as being sites forthe sequence-specific interaction of one or more transcription factors,frequently taking the form of direct protein-DNA binding. Typically,transcription factor binding sites can be identified by DNAfootprinting, gel mobility shift assays, and the like, and/or can bepredicted on the basis of known consensus sequence motifs, or by othermethods known to those of skill in the art.

As used herein, the term “operably linked” refers to a linkage of two ormore polynucleotides or two or more nucleic acid sequences in afunctional relationship. A nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. For instance, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the coding sequence.Operably linked means that the DNA sequences being linked are typicallycontiguous and, where necessary to join two protein coding regions,contiguous and in reading frame. However, since enhancers generallyfunction when separated from the promoter by several kilobases andintronic sequences may be of variable lengths, some polynucleotideelements may be operably linked but not contiguous.

“Transcriptional unit” refers to a polynucleotide sequence thatcomprises at least a first structural gene operably linked to at least afirst cis-acting promoter sequence and optionally linked operably to oneor more other cis-acting nucleic acid sequences necessary for efficienttranscription of the structural gene sequences, and at least a firstdistal regulatory element as may be required for the appropriatetissue-specific and developmental transcription of the structural genesequence operably positioned under the control of the promoter and/orenhancer elements, as well as any additional cis sequences that arenecessary for efficient transcription and translation (e.g.,polyadenylation site(s), mRNA stability controlling sequence(s), etc.

The term “substantially complementary,” when used to define either aminoacid or nucleic acid sequences, means that a particular subjectsequence, for example, an oligonucleotide sequence, is substantiallycomplementary to all or a portion of the selected sequence, and thuswill specifically bind to a portion of an mRNA encoding the selectedsequence. As such, typically the sequences will be highly complementaryto the mRNA “target” sequence, and will have no more than 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 base mismatches throughout the complementary portionof the sequence. In many instances, it may be desirable for thesequences to be exact matches, i.e. be completely complementary to thesequence to which the oligonucleotide specifically binds, and thereforehave zero mismatches along the complementary stretch. As such, highlycomplementary sequences will typically bind quite specifically to thetarget sequence region of the mRNA and will therefore be highlyefficient in reducing, and/or even inhibiting the translation of thetarget mRNA sequence into polypeptide product.

Substantially complementary oligonucleotide sequences will be greaterthan about 80 percent complementary (or ‘% exact-match’) to thecorresponding mRNA target sequence to which the oligonucleotidespecifically binds, and will, more preferably be greater than about 85percent complementary to the corresponding mRNA target sequence to whichthe oligonucleotide specifically binds. In certain aspects, as describedabove, it will be desirable to have even more substantiallycomplementary oligonucleotide sequences for use in the practice of theinvention, and in such instances, the oligonucleotide sequences will begreater than about 90 percent complementary to the corresponding mRNAtarget sequence to which the oligonucleotide specifically binds, and mayin certain embodiments be greater than about 95 percent complementary tothe corresponding mRNA target sequence to which the oligonucleotidespecifically binds, and even up to and including 96%, 97%, 98%, 99%, andeven 100% exact match complementary to all or a portion of the targetmRNA to which the designed oligonucleotide specifically binds.

Percent similarity or percent complementary of any of the disclosedsequences may be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0, available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch (1970).Briefly, the GAP program defines similarity as the number of alignedsymbols (i.e., nucleotides or amino acids) that are similar, divided bythe total number of symbols in the shorter of the two sequences. Thepreferred default parameters for the GAP program include: (1) a unarycomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and anadditional 0.10 penalty for each symbol in each gap; and (3) no penaltyfor end gaps.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Improved rAAV Vectors Having Genetic Modifications in SpecificCapsid Proteins

Given advances in purification methods for rAAV2, the requirements ofthe individual capsid protein species in rAAV2 particle formation werereexamined in the context of designing a novel rAAV2 production systemthat would allow for the modification of a specific capsid protein inregions of capsid sequence overlap. Currently, highly purified andconcentrated preparations of rAAV2 particles are possible from twoplasmid-based production systems. These systems differ in that onesystem supplies the necessary adenovirus helper functions and AAV repand cap genes from one plasmid (pDG), while the other uses two plasmidsto supply these proteins (pIM45 and pXX6). These constructs aretransfected into an appropriate cell type along with a constructcontaining a transgene expression cassette flanked by the AAV terminalrepeats (e.g., pTR-UF5). This example describes an rAAV2 productionsystem based on modifications of the triple plasmid transfection method.In this system, the expression of a specific capsid protein isrestricted to one pIM45 plasmid and complemented in trans with theremaining two capsid proteins expressed from a second pIM45 plasmid.This approach maintains expression of the capsid proteins in theirgenomic context while providing a platform for the genetic modificationof a specific capsid protein or two of the capsid proteins across theirentire coding sequence. Missense mutation of the capsid proteins' startcodons generated pIM45 plasmids that express a single capsid protein:pIM45-VP1, pIM45-VP2 (ACG or ATG start codon), and pIM45-VP3. Suchplasmids can be complemented with plasmids expressing the remaining 2capsid proteins (pIM45-VP2,3, pIM45-VP1,3, and pIM45-VP1,2 (ACG or ATGstart codon), respectively) in order to produce viable rAAV2 vectors.Using the system's plasmid components individually, a reevaluation ofcapsid protein requirements for the production of rAAV2 particlesrevealed that viable rAAV2-like particles are produced as long as theVP3 protein is present (VP1+2+3, VP1+3, VP2+3, and VP3 only). Focusingon large peptide insertions in the VP1 and VP2 proteins without alteringthe critical VP3 protein, the utility of this system is demonstratedthrough the production of viable rAAV2 particles containing 8-, 15-, and29-kDa proteins inserted immediately following amino acid 138 in bothVP1 and VP2 proteins or in VP2 protein alone. Finally, rAAV2-likeparticles can be produced with altered capsid protein stoichiometry ifVP2 is significantly over expressed.

Construction of rAAV2 Capsid Mutant Plasmids that Express 2 CapsidProteins

To isolate the expression of a specific capsid protein to one pIM45plasmid and the remaining two capsid proteins to a second pIM45 plasmid,missense mutation of the AAV2 cap ORF start codons was employed aspreviously described. Using site-directed mutagenesis of a pIM45template, the VP1 start codon was mutated to leucine to generate theconstruct, pIM45-VP2,3, the VP2 start codon to alanine to generate theconstruct, pIM45-VP1,3, and the VP3 start codon to leucine to generatethe construct, pIM45-VP1,2 (FIG. 1A-1 and FIG. 1A-2). Western blottinganalysis of capsid protein expression in whole cell lysates 48 hrs posttransfection of 293 cells with these plasmids in the presence of Ad5(MOI=10) was carried out using the B1 antibody which recognizes allthree capsid proteins (FIG. 1A-1 and FIG. 1A-2). As previously reported,the expression of VP1 and VP2 could be eliminated by missense mutationof their start codons (FIG. 1A-1, lanes 2 and 3), and, in contrast,mutation of the VP3 start codon resulted in expression of a smallerVP3-like fragment (VP3a) (FIG. 1A-1, lane 4). Since this construct didnot eliminate all VP3-like proteins it was renamed, pIM45-M203L. In thebaculovirus study of AAV particle assembly, it was suggested thatmutation of the VP3 start codon allows translational initiation to occurdownstream at the next available ATG codon with correct Kozak sequences.While no additional ATG codons are found between the VP1 start codon andthe start of VP3, an examination of the VP3 capsid revealed that nineadditional ATG codons are present (amino acid positions 211, 235, 371,402, 434, 523, 558, 604, and 634). Of these methionines, only those atamino acid position 211, 235, 523, 558, and 604 are in a context that ispredicted favorable by Kozak. Since the VP3a fragment is slightlysmaller than wildtype VP3, the contribution of continued read throughtranslational initiation to the appearance of the VP3a fragment wasexamined by mutating the next two available ATG codons (M211 and M235)on a pIM45-M203L template yielding the plasmids, pIM45-M203L,pIM45-M203,211L and pIM45-M203,211,235L (FIG. 2A). Western blottinganalysis of capsid protein expression in whole cell lysates 48 hrs posttransfection of 293 cells in the presence of Ad5 (MOI=10) revealed thattranslational initiation could occur at both these ATG codons. FIG. 1B-1and FIG. 1B-2 (lane 2) again demonstrates the formation of VP3afollowing the mutation M203L. Combined mutation of M203 and M211 allowedless robust expression of a second still shorter VP3-like fragment(VP3b, FIG. 1B-1 and FIG. 1B-2, lane 3). Subsequent mutation of M235 inthe pIM45-M203,211L background led to disappearance of this VP3bfragment generating pIM45-VP1,2 (FIG. 1B-1, lane 4). Collectively, whilemissense mutagenesis of the VP1 start codon does not alter the sequenceof the VP2 and VP3 protein expressed (pIM45-VP2,3, M1L), mutation of theVP2 start codon results in one point mutation in the expressed VP1protein (pIM45-VP1,3, T138A), and elimination of all VP3-like proteinsresults in three mutations in the remaining VP1 and VP2 proteins(pIM45-VP1,2, M203,211,235L).

An alternative method has been reported for eliminating VP3 expressionthat limits mutation of remaining capsid sequences to one point mutationin the VP2 start codon. Changing the VP2 start codon from ACG to ATGresults in loss of VP3 expression (pIM45-VP1,2A) with one point mutationin both the VP1 and VP2 proteins (T138M). Presumably, this stronger VP2start codon prevents efficient translational initiation at thedownstream VP3 start codon. The VP2 start codon was mutated to ATG on apIM45 template [pIM45-VP1,2A (FIG. 1C-1 and FIG. 1C-2)] as analternative means of eliminating VP3 protein (while maximizing VP2expression). As expected, Western blotting analysis of capsid proteinexpression in whole cell lysates 48 hrs post transfection of 293 cellsin the presence of Ad5 (MOI=10) with pIM45-VP1,2A showed normal levelsof VP1 protein produced, with significantly increased expression of VP2protein (FIG. 1C-2, lane 2).

Construction of rAAV2 Capsid Plasmid Mutants that Express a SingleCapsid Protein

To complete the complementary pIM45 capsid groups, pIM45 plasmids thatexpress a single capsid protein were generated next. Employing the samemissense mutations described above on templates that now only expresstwo capsid proteins, the plasmids, pIM45-VP1, pIM45-VP2, pIM45-VP2A, andpIM45-VP3 (FIG. 2A and FIG. 2B) were also generated. pIM45-VP1 has theVP2 start codon mutated to alanine and M203, M211, and M235 mutated to Lin the expressed VP1 protein. pIM45-VP2 has the VP1 start codon mutatedto leucine and M203, M211, and M235 mutated to L. The expressed VP2protein contains only M203, M211, and M235 mutations. pIM45-VP3 has theVP1 start codon mutated to leucine and the VP2 start codon mutated toalanine. Like all VP3 protein in these complementary groups, the VP3coding sequence is not mutated. Finally, pIM45-VP2A has the VP1 startcodon mutated to leucine and the VP2 start codon mutated to methionineresulting in the single T138M modification of the VP2 protein beingexpressed. Western blotting analysis of capsid protein expression inwhole cell lysates 48 hr post transfection of 293 cells with pIM45-VP1,pIM45-VP2, pIM45-VP2A, and pIM45-VP3 in the presence of Ad5 (MOI=10)demonstrated that a single capsid protein could be expressed from thepIM45 cap ORF (FIG. 2A and FIG. 2B) and completed the catalogue ofplasmids required of a system for further genetic manipulation of aspecific capsid protein across its entire coding sequence.

VP3 N-Terminal M203 and M211 are Critical for AAV Particle Formation

As control experiments for the production of AAV particles from thecomplementary groups of single and double capsid expressing pIM45plasmids, particle production was examined from the individual plasmidsdescribed. Since VP3 protein makes up the bulk of the particle, andmutagenesis studies have indicated that the N-terminal region of VP3 isimportant for AAV particle formation, the effects of the three mutationsrequired to eliminate VP3 expression (M203,211,235L) were investigatedon the recovery of rAAV particles following standard production andpurification protocols. The plasmids pIM45-M203L, pIM45-M211L,pIM45-M235L, and pIM45M-203,211,235L were cotransfected separately withpTR-UF5 and pXX6 in a 1:1:8 molar ratio in 293 cells and 72 hrs laterthe cells were harvested and particles were purified as previouslyreported. Western blotting of capsid protein expression and dot blotanalysis of genome containing particles was carried out on the mutantvirus preparations (FIG. 3A-1 and FIG. 3A-2). No particles wererecovered from pIM45-M203L (lane 2) indicating that the combination ofVP1, VP2, and VP3a does not able form a stable AAV particle. Equallyimportant in the formation of the particle is M211 (lane 3), as thismutation also prevented particle recovery. Whether it is the M211L inVP1, VP2, or VP3 that leads to this defective phenotype is unclear. Thisissue is addressed infra when pIM45-VP1,2 is complemented with pIM45-VP3to produce AAV particles (FIG. 4, #5). Finally, particles were obtainedfrom pIM45-M235L (FIG. 3A-1 and FIG. 3A-2, lane 4) that package DNAefficiently.

AAV-Like Particles can be Produced that Lack VP10R VP2 Protein

While the effect of mutating the individual capsid start codons on theformation of infectious AAV particles has been reported, given theimprovements in AAV2 production and purification methods, controlexperiments were performed to reexamine the role of each capsid proteinin the formation of the AAV2 particle capable of binding heparin. Firstexamined were the effects of the elimination of one capsid protein onAAV2 particle recovery. pIM45-VP2,3; pIM45-VP1,3, pIM45-VP1,2; andpIM45-VP1,2A; were transfected separately into 293 cells with pTR-UF5and pXX6 in a 1:1:8 molar ratio and 72 hrs later the cells wereharvested and particles were purified as previously reported. Westernblotting, A20 ELISA, and dot blot analysis of these virus preparationswere carried out (FIG. 3B-1 and FIG. 3B-2) and, in agreement withprevious reports, the elimination of the VP1 protein (pIM45-VP2,3)resulted in the production of an AAV-like particle that packaged genomesefficiently (lane 4). Surprisingly, in contrast with the initial reportmapping the capsid start codons, transfection of the pIM45-VP1,3 plasmidresulted in the purification of an AAV-like particle capable ofpackaging genomes efficiently composed of only VP1 and VP3 (lane 3) thathad only a modest decrease in infectivity compared to particlescontaining all three capsid proteins (two-fold decrease). Finally,regardless if VP2 is overexpressed, particles composed of only VP1 andVP2 were not recovered (lane 2).

AAV-Like Particles can be Produced Composed Only of VP3 Capsid Proteins

As with the pIM45 plasmids that express two capsid proteins, the abilityof a single capsid protein to form an AAV-like particle was tested.pIM45-VP1, pIM45-VP2, pIM45-VP2A, and pIM45-VP3 were transfectedseparately into 293 cells with pTR-UF5 and pXX6 in a 1:1:8 molar ratioand harvested cells 72 hrs later and purified particles as previouslydescribed. Western blotting of capsid proteins, A20 ELISA, and dot blotanalysis of virus preparations were carried out with no detectableAAV-like particles obtained from pIM45-VP1, pIM45-VP2, or pIM45-VP2A(FIG. 3C-1 and FIG. 3C-2, lanes 2 and 3). Interestingly, like a recentinsertional mutagenesis study of the cap ORF, an AAV-like particlecomposed exclusively of VP3 protein was purified (lane 3). Like theVP2,3 AAV-like particle, this particle had a significantly lowerinfectious phenotype.

rAAV Particles with all Three Capsid Proteins can be Produced fromCapsid Complementation Groups

Given the results of the control experiments, the ability to recoverrAAV2 particles containing all three capsid proteins followingtransfection of two complementary pIM45 plasmids was tested (FIG. 4). Tocontrol for twice the Rep expression resulting from two pIM45 plasmids,an additional plasmid was constructed, pIM45-VP0, that expresses nocapsid proteins as a result of 5 point mutations (M1L, T138A,M203,211,235L). Complementary group VP0 (FIG. 4, #1) includes pIM45 andpIM45-VP0, group VP1 includes pIM45-VP1 and pIM45-VP2,3 (FIG. 4, #2),group VP2 includes pIM45-VP2 and pIM45-VP1,3 (FIG. 4, #3), group VP2Aincludes pIM45-VP2A and pIM45-VP1,3 (FIG. 4, #4), and group VP3 includespIM45-VP3 and pIM45-VP1,2 (FIG. 4, #5). Western blotting of capsidproteins, A20 ELISA, and dot blot analysis of virus preparations werecarried out following transfection of the individual groups into 293cells with pTR-UF5 and pXX6 in a 1:1:8 molar ratio. 72 hrs posttransfection the cells were harvested and particles were purified aspreviously described. Infectious rAAV particles containing all threecapsid proteins with similar yields were recovered (FIG. 5A-1 and FIG.5A-2). Interestingly, the VP2A group resulted in the formation ofparticles with an apparent alteration of capsid protein stoichiometryand lower infectivity compared to other groups (lane 4). Thecharacteristics of this group suggest that this preparation may containa single unique particle that is defective per se or alternatively twoparticles may be assembled containing all three capsid proteins atnormal levels and a defective interfering particle composed of VP2 andVP3 proteins with altered stoichiometry. Cotransfection of thepIM45-VP2A and pIM45-VP3 plasmid should yield a particle with an alteredVP2:VP3 ratio if such a defective interfering particle contributes tothe low titer of this group. Indeed, an AAV-like particle with anoverrepresentation of VP2 protein was purified that resembled the VP2,3and VP3 only particles with respect to infectivity (FIG. 5B-1 and FIG.5B-2, lane 4).

Production of AAV Particles with Insertions in the VP1/VP2 OverlapRegion

Since the VP1/VP2 overlap region has been shown to be on the surface ofthe particle and flexible in the acceptance of targeting epitopes, theability of this region to accept larger insertions was examined.Presumably, large insertions in the VP3 protein would decrease onessuccess in obtaining a particle due to steric hindrances in assemblingthe 60 modified capsid subunits. Sixty ligands were considered excessivewhen inserting large molecules into the AAV particle, so the strategyemployed was to focus larger insertions to VP1 and/or VP2 proteins.Large insertions in both VP1 and VP2 protein immediately after aminoacid 138 may have less steric constraints but may produce particles withdefective trafficking due to the juxtaposition of a large insertion tothe putative phospholipase motif in VP1 protein. Also, since VP1,essentially an N-terminal fusion of 137 amino acids to VP2, and a CD 34sc antibody VP2 protein fusion are readily incorporated into an AAVparticle, insertion of large epitopes only at the N-terminus of VP2 mayhave advantages. Notably, genetic modification of the VP2 proteinexclusively has not been accomplished from within a pIM45 based AAVproduction scheme. To address the ability to insert large peptidesequences in the VP1/VP2 overlap region of the cap ORF, directionalcloning sites were generated immediately after amino acid 138 inplasmids that express VP1 and VP2 or VP2 only (FIG. 6A). The choice ofEagI/MluI restriction sites resulted in two amino acids insertions oneither side of further inserted sequence (RP/RT). pIM45-VP1,2A andpIM45-VP2A were chosen as templates for engineering EagI/MluIrestriction sites immediately after amino acid 138 in VP1 and VP2 or inVP2 only (pIM45VP1,2AEM138, pIM45-VP2AEM138, FIG. 6A). Templates withVP2 protein over expression were used to help ensure thatgenetically-modified VP2 would be present at sufficient levels forassembly. The pIM45-VP1,2AEM138 plasmid was complemented with pIM45-VP3,while the pIM45-VP2AEM138 plasmid was complemented with pIM45-VP1,3 forthe production of rAAV particles carrying insertion sequences. Insertionof the coding sequence for leptin and GFP was in these plasmidsgenerated pIM45-VP1,2Alep, pIM45-VP2Alep, pIM45-VP1,2AGFP, andpIM45-VP2AGFP. These plasmids and their complements were transfectedwith in 293 cells in the presence of pTR-UF5 (leptin-insertions) orpTR-dsRFP (GFP) and pXX6 in a 1:1:8 molar ratio. 72 hrs posttransfection the cells were harvested and virus particles were purifiedas previously described. Western blotting, A20 ELISA, and dot blotanalysis were carried out on the virus preparations (FIG. 6B-1, FIG.6B-2, FIG. 6C-1 and FIG. 6C-2). For the leptin-inserted viruspreparations successful insertion in both VP1 and VP2 or VP2 only waspossible in the purified particle, but GFP-insertion in the purifiedparticle was only possible in the VP1 protein (VP2-GFP was excluded inboth cases).

Discussion

This example increases the flexibility in probing the surface of theparticle by isolating the expression of a given capsid protein to aseparate plasmid. Such an approach allows for manipulation of thiscapsid protein only within the produced particle and allows forretesting regions of capsid overlap for the acceptance of sequencemodification. Alternatively, the system also allows for the modificationof only two of the capsid proteins while leaving the third proteinunmodified. Using the missense mutation of capsid start codons togenerate all required plasmids, characterization of the catalogue ofplasmids required for this system yielded interesting results concerningthe role of each capsid protein in the assembly of AAV-like particles.

Elimination of VP3-like fragments illustrates importance of VP3 Nterminus, as particles with these mutations in the VP1 and VP2 proteinswere recovered following complementation of pIM45-VP1,2 with pIM45-VP3.Evidence that ability to modify individual capsid proteins in regions ofoverlap may allow production of particles that were defective forproduction when mutations are in all three proteins. Increases theflexibility in manipulation of the particle for targeting purposes.Recently, isolation of the expression of a C-terminal modified VP3separately allowed for modification of the c-terminus of VP3 with histag and production of viable recombinant virus follow nickelchromatography. Like the study involving the VP3-6×His tag where themodified capsid protein was isolated and VP1 and VP2 did not carry theinsertion, lethal mutations in the overlapping N-terminus region of VP3(M203,211) resulted in particles from complementation group 3 when thesemutations were only in VP1 and VP2 with normal VP3.

The present system allows for complementation and recovery of rAAV2particles with all capsid proteins present. Since it allows for thegenetic modification of only one or two of the capsid proteins, it canalso be used for studies of previously reported lethal mutations inoverlapping capsid sequences to see if mutations at the same positionsin fewer capsid proteins rescue the position for particle manipulation.Important genetic modification would include insertion of geneticsequence for retargeting the virus, purification of the virus,monitoring of the virus particle following infection, or presentation ofimmunogenic epitopes on the surface of the virus particle.

Insertions of large peptides (leptin and GFP) into the overlappingregion of VP1 and VP2 resulted in the purification of virus likeparticles carrying these insertions. This required preliminary isolationof the expression of VP1 and VP2 (pIM45-VP1,2A) or VP2 only (pIM45-VP2A)to a separate plasmid followed by insertion of peptide sequences afteramino acid 138 allowed for the production of peptide inserted AAV-likeparticles following complementation with pIM45-VP3 or pIM45-VP1,3. Thisexample is the first report of the purification of an AAV-like particlecontaining a mutation in the VP2 protein exclusively. Estimated similarstoichiometry of capsid proteins in particle. Retain ability to packagegenomes, bind A20, and are infectious as they retain native tropism dueto intact heparin binding motif. VP2 overexpression may have ensured theinclusion of modified VP2 protein large insertions with VP2 acg startcodon produced significantly less modified VP2 proteins.

Example 2 Heparin Sulfate Binding Motif in AAV2 Capsid Proteins Requiredfor Native Tropism

In this example, charged-to-alanine substitution mutants were made toanalyze the effects of single and combinatorial mutations in the capsidgene. New point mutants that result in assembly, packaging, and receptorbinding deficiencies have been discovered. Importantly, five aminoacids, arginines 484, 487, 585, and 588, and one lysine at position 532have been identified that appear to mediate the natural affinity of AAVfor HSPG. Those observations contribute to the current map of the AAVcapsid and provide a reagent for the discovery of novel, heparinindependent targeting ligands.

Materials and Methods

Plasmids.

Plasmid pIM45 (previously called pIM29-45) contains the Rep and Capcoding sequences from AAV with expression controlled by their naturalpromoters (McCarty et al., 1991). It was used as the parent template forconstruction of all the AAV2 mutant vectors.

Plasmid pXX6 supplies the adenovirus helper gene products in trans toallow rAAV production in an adenovirus free environment (Xiao et al.,1998). Plasmid pTR2-UF5 supplies the recombinant AAV DNA to be packaged.It contains a cytomegalovirus promoter driving expression of a greenfluorescent protein (GFP) reporter gene flanked by AAV2 terminal repeats(Klein et al., 1998). Plasmid pTR5-UF11 was constructed using anexpression cassette consisting of a strong constitutive CBA promoter (Xuet al., 2001), GFP reporter gene (Zolotukhin et al., 1996), woodchuckhepatitis virus posttranscriptional regulatory element WPRE (Donello etal., 1998) and bovine growth hormone gene polyadenylation signal. Thecassette was assembled using standard molecular biology techniques andsubstituted for the lacZ cassette in the plasmid backbone pAAV5RnlacZcontaining AAV5 terminal repeats (Chiorini et al., 1999). PlasmidspXYZ1, pXYZ5 contain the AAV1 and AAV5 Cap coding sequences,respectively, in addition to AAV2 Rep coding sequence with an ACG startcodon under control of the AAV2 p5 promoter (Zolotukhin et al., 2002).Plasmid pAAV5-2 contains the AAV5 nucleotides 260 to 4448 withoutterminal repeats (Chiorini et al., 1999).

Construction of Mutant Capsid Plasmids.

Quickchange site directed mutagenesis (Stratagene) was performed onplasmid pIM45 as per the manufacturer's instructions. For each AAV2mutant, two complementary PCR primers that contained alanine or lysinesubstitutions in addition to a silent change for restrictionendonuclease screening purposes were used to introduce changes intopIM45. For construction of AAV5-HS, pAAV5-2 was used as the parentaltemplate. Sequences for the oligonucleotides used are available uponrequest. PCR products were digested with DpnI to remove methylatedtemplate DNA, phenol:cholorform:isoamyl alcohol (25:24:1) extracted,ethanol precipitated, and transformed into electrocompetent JM109 cells.Miniprep DNA was extracted from overnight LB/amp cultures and screenedwith the appropriate restriction enzyme. All mutants were sequencedprior to use. Transfection quality plasmid DNA was produced by standardalkaline lysis method of a 1-liter TB culture followed by PEGprecipitation and cesium chloride gradient purification.

Cell Culture

Human embryonic kidney 293's and cervical carcinoma HeLa C12's, a giftfrom Dr. Phil Johnson (Clark et al., 1996) were grown in DulbeccoModified Eagle Medium (Gibco-BRL) supplemented with 100 U/mL penicillin,100 U/mL streptomycin, 10% bovine calf serum, sodium pyruvate andL-glutamine. Cells were incubated at 37° C. in a 5% CO₂ atmosphere.

Production of rAAV2 Particles

To produce AAV2 virions, low passage 293's were seeded so that they wereapproximately 75% confluent at transfection time. A triple plasmidtransfection protocol (Xiao et al., 1998) was followed that includedpIM45 to supply Rep and mutated capsid genes, pTR2-UF5 (Klein et al.,1998) to supply recombinant DNA with AAV2 terminal repeats and a CMVdriven GFP reporter gene, and pXX6 (Xiao et al., 1998) to supply theadenovirus helper functions in trans. A total of 60 μg of plasmid DNA ina 1:1:1 molar ratio was transfected by lipofectamine (Invitrogen).

To produce pseudotyped rAAV1 and rAAV5 particles, a total of 60 μg ofpXYZ1 or pXYZ5 (Zolotukhin et al., 2002) was co-transfected withpTR2-UF5 plasmid DNA in a 1:1 molar ratio as above. To produce rAAV5 andrAAV5-HS virions, a total of 60 μg of pAAV5 or pAAV5-HS wasco-transfected with pTR5-UF 11.

Purification of rAAV has been described previously (Zolotukhin et al.,1999; Zolotukhin et al., 2002). Briefly, 72 hrs after transfection,cells were harvested and the pellets were resuspended in lysis buffer(0.15 M NaCl, 50 mM Tris-Cl pH=8.5). Virus was released by three cyclesof freezing and thawing. Benzonase (Sigma) was added to the cell lysateto a final concentration of 140 U/mL and incubated at 37° C. for 30 min.Cell debris was pelleted by centrifugation at 3,700×g for 30 min and thesupernatant was loaded onto a 15%-25%-40%-60% iodixanol(5,5′[2-hydroxy-1,3-propanediyl)bis(acetyl-amino)]bis[N,N′-bis(2,3dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide]step gradient (Nycomed). The 40% fraction was collected aftercentrifugation at 69,000×g for 1 hr and stored at −80° C. until furtheruse.

Virus Titer Determination

To determine the concentration of intact capsid particles, the A20 ELIZA(American Research Bioproducts) was used. The A20 antibody detectsintact, fully assembled particles, both full and empty (Wistuba et al.,1995). Iodixinal purified stocks were serially diluted and processed bythe manufacturer's recommended protocol. Only readings within the linearrange of the kit standard were used.

To determine the concentration of DNA-containing particles, real-time(RT)-PCR™ was performed using a Perkin Elmer-Applied Biosystems (FosterCity, Calif.) Prism 7700 sequence detector system. Equal volumes ofiodixanol purified virus stocks were treated with 600 U/mL benzonase in50 mM Tris-CL pH=7.5, 10 mM MgCl₂, 10 mM CaCl₂ at 37° C. for 30 mM. 280U/mL Proteinase K was added to reactions adjusted to 10 mM EDTA and 5%SDS, and then incubated at 37° C. for 30 mM. Reactions were extractedwith phenol/chloroform/isoamyl-alcohol (25:24:1) and undigested DNA wasprecipitated overnight with ethanol and glycogen carrier. PrecipitatedDNA pellets were resuspended in 100 μl of water. Five μl was used forRT-PCR™ analysis in a reaction mixture that included 900 nM each of GFPforward (5′-TTCAAAGATGACGGGAACTACAA-3′) (SEQ ID NO:3) and reverse(5′-TCAATGCCCTTCAGCTCGAT-3′) (SEQ ID NO:4) primers, 250 nM Taqman® probe(5′-6-FAM-CCCGCGCTGAAGTCAAGTTCGAAG-TAMRA-3′) (SEQ ID NO:5), 1× Taqman®universal PCR master mix in a total volume of 50 pt. Cycling parameterswere 1 cycle each of 50° C., 5 min, and 95° C., 10 min, followed by 40cycles of 95° C., 15 sec and 60° C., 1 min. Only values within thelinear portion of a standard curve having a coefficient of linearitygreater than 0.98 were accepted. The average RT-PCR™ titer wascalculated from virus preparations assayed three times.

To determine the infectious titer of the wt and mutant virus stocks, agreen cell assay (GCA) was performed essentially as previously described(Zolotukhin et al., 1999). Briefly, HeLa C12 cells were seeded in a96-well plate so that they were approximately 75% confluent at infectiontime. Cells were infected with 10-fold serial dilutions of iodixanolpurified mutant viruses and Ad5 at a constant multiplicity of infection(MOI)=10. Cells were incubated at 37° C. in a 5% CO₂ atmosphere for 24hrs and examined by fluorescence microscopy. The average GCA titer wascalculated by averaging the number of green cells counted in individualwells from two or three virus preparations assayed three times. Particleto infectivity ratios were calculated by dividing the average RT-PCR™titer by the average GCA titer. In some figures, this number wasexpressed as a log₁₀ value with rAAV2 arbitrarily set to one.

In Vitro Heparin Binding Assay

Bio-Rad microspin columns were treated with silicon dioxide to minimizenon-specific binding of the virus to the column wall. A 500 μLheparin-agarose (Sigma H-6508) gravity column was prepared by washingwith 3 column volumes each of 1×TD (137 nM NaCl, 15 mM KCl, 10 mMNa₂PO₄, 5 mM MgCl₂, 2 mM KH₂PO₄, pH=7.4), 1×TD+2 M NaCl and 1×TD.Approximately equal numbers of virus particles were added to 1×TD to afinal volume of 600 μL and loaded onto the column. The column was washedwith 7 column volumes of 1×TD. Bound virus was eluted with 1×TD+2 MNaCl. The entire volume of the flow through, wash, and eluate fractionswere pooled separately, denatured by boiling in SDS, and slot blottedonto nitrocellulose for immunoblot analysis. The membrane (Osmonics) wasblocked in PBS/0.05% Tween-20+5% dry milk, and incubated with B1antibody (Wistuba et al., 1997) at a 1:3000 dilution for 18 hrs at 4° C.Anti-mouse IgG-horse radish peroxidase was used to detect bands byenhanced chemiluminesence (Amersham-Pharmacia).

Fluorescence Activated Cell Sorting (FACS)

HeLa C12 cells were seeded in 6 well plates so that they wereapproximately 75% confluent at infection time. Cells were infected withan rAAV MOI=500 based on the genomic titer as determined by DNA dot blotassay (Zolotukhin et al., 1999). Adenovirus type-5 was used at an MOI=10plaque forming units (pfu). Twenty-four hrs postinfection, cells werewashed, trypsinized, and fixed in 2% paraformaldyhede. FACS analysis forGFP expression was done in the ICBR Flow Cytometry lab of the Universityof Florida on a Becton-Dickinson FACScan.

Cell Attachment Assay

10⁶ Hela C12 cells were infected with rAAV2 at a genome containingparticle MOI=100 or R585A/R588A at an MOI=1000, as determined byRT-PCR™. Cells were incubated at 37° C. in a 5% CO₂ atmosphere untilharvesting. At indicated time points, the infection media was removedand saved and the cells were washed four times with PBS before beingscraped. Low-molecular-weight DNA from the infection media and the cellpellet was extracted (Hirt, 1967). DNA pellets were resuspended in 0.2 MNaOH, incubated at 37° C. for 20 min, and slot blotted ontonitrocellulose. DNA was UV cross-linked to the nitrocellulose and probedat 65° C. for 18 hrs with [α-³²P]-dATP labeled GFP probe inhybridization buffer (7% SDS, 10 mM EDTA and 0.5 M Na₂HPO₄). Membraneswere washed twice in 2×SSC/0.1% SDS, 0.2×SSC/0.1% SDS, 0.1×SSC/0.1% SDS,and rinsed with water. The membranes were then exposed to film andquantitated using a BAS-1000 phosphor imager (Fuji).

Results

Selection and Generation of AAV Mutants

A considerable body of information regarding the determinants ofHS-protein interactions suggests that their association is driven mainlyby electrostatic attraction between acidic sulfate groups on thepolysaccharide and basic R-groups on amino acids in the target protein(Hermens et al., 1999; Hileman et al., 1998). It was hypothesized thatsimilar electrostatic interactions would govern HSPG-AAV2 association.In order to evaluate the role of particular amino acids in receptorbinding, a panel of mutants was generated by site directed mutagenesisof selected residues. The selection was confined primarily to basicamino acids (His, Lys, Arg) in VP3 as AAV-like particles composed onlyof VP3 proteins have been purified by heparin affinity chromatography.Any basic amino acid substitution mutant that previously haddemonstrated capsid instability or efficient purification by heparinaffinity chromatography (Wu et al., 2000) was excluded from the pool ofmutants.

Seven AAV serotypes have been reported (Bantel Schaal and zur Hausen,1984; Gao et al., 2002; Hoggan et al., 1996; Parks et al., 1967;Rutledge et al., 1998). Several groups have shown that rAAV2 and rAAV3bind efficiently to heparin sulfate (Rabinowitz et al., 2002; Shi etal., 2001; Wu et al., 2000). A single report concerning rAAV1 suggeststhat it binds with low affinity, if at all, to heparin (Rabinowitz etal., 2002). In contrast, rAAV4 and rAAV5 do not bind heparin and insteadrecognize 2,3 O-linked and 2,6 N-linked sialic acid moieties (Kaludov etal., 2001). Indeed, this may account for their different cellulartropisms. It was reasoned that residues conserved among all fiveserotypes were probably not participating directly in receptordiscrimination and binding and were excluded from further consideration.Additionally, a number of charge-to-alanine substitution mutants in theAAV capsid had been identified, and these had been characterized fortheir ability to bind heparin sulfate columns (Wu et al., 2000) andamino acid positions that did not affect heparin binding or had beenshown to be assembly mutants were excluded from further study. Using aClustal W algorithm, a sequence alignment of capsid proteins fromserotypes 1-5 was generated, and 9 basic residues in AAV2 that wereconserved in AAV3 and/or AAV1 but were uncharged or acidic in AAV4 andAAV5 were identified that had not previously been tested forheparin-agarose binding (Table 4). In addition to these 9 amino acids,Wu et al. (2000) described a virus deficient for heparin binding withalanine substitution mutations at positions 585, 587, and 588. Finally,during the course of these studies, the atomic structure of AAV2 wassolved (Xie et al., 2002) and suggested that residues 484, 513, and 532might participate in a heparin-binding pocket as they were located closeto residues 585, 587, and 588. These six extra residues were alsoincluded to complete the mutant panel (Table 4).

TABLE 4 RESIDUES CHOSEN FOR MUTAGENESIS AAV Serotype^(b) VP Residue^(a)2 3 1 4 5 358 H H H Q T 447 R R R S S 459 R R D T G 484 R R R K R 487 RR R G G 509 H H H T E 513 R R R R A 526 H H H A N 527 K K K G N 532 K KK K N 544 K K K P S 566 R R K A Q 585 R S S S S 587 N N S S T 588 R T TN T ^(a)Residues selected for mutagenesis were generated by a sequencealignment of the VP1 capsid protein from each serotype using the ClustalW algorithm (Vector NTi 5.2, Informax). ^(b)Amino acids are representedby their one letter abbreviation. Blue letters represent positivelycharged, basic amino acids. Red letters represent any other amino acid.

Mutant Virus Production and Physical Characterization

A series of single and combinatorial capsid mutants were generated fromthe pool of candidate residues in the AAV2 capsid gene (Table 4). Todesignate the mutant viruses, the number of the mutated amino acid basedon its position in VP1 was used. Iodixanol purified virus stocks werechecked by western blot using the monoclonal antibody B1. The B1antibody recognizes a linear epitope in the extreme carboxyl terminus ofall three VP proteins from AAV serotypes 1, 2, 3 and 5 (Rabinowitz etal., 2002; Wobus et al., 2000). With the exception of H358A, capsidproteins were detected in all virus stocks (FIG. 7). To confirm thatassembled capsids, rather than subunits or assembly intermediates, hadbeen purified, the particle concentration was measured with an A20antibody ELISA (Table 5). The A20 antibody recognizes a structuralepitope that is found only on assembled capsids with or without packagedDNA (Grimm et al., 1998). Although there was some variability betweenstocks due to different transfection efficiencies and purificationrecoveries, only the H358A mutant was negative by A20 ELISA assay.Excluding H358A, a particle concentration range was determined thatspanned 1.5 logs and correlated reasonably well with the B1 antibodyresults (FIG. 7; Table 5). Several possibilities may account for thisrange of particle titers, including that capsid subunits containingthese mutations (i) form intact particles inefficiently, (ii) areunstable during purification or (iii) formed a particle with a partiallydisrupted A20 epitope. Since none of these mutations fell within theantigenic regions that have been mapped for A20 (Wobus et al., 2000),these results suggested that the A20 epitope had probably not beenmodified but rather the stability or assembly of some of the mutants wasaltered so that fewer particles were recovered after iodixanolcentrifugation (FIG. 7; Table 5).

TABLE 5 TITERS AND HEPARIN BINDING PROPERTIES OF MUTANTS InfectiousParticle Titer^(b) Titer^(c) Particle-to Heparin Empty/ Mutant Virus^(a)A20/mL Genome/mL (IU/mL) infectivity Binding^(e) Full^(g) rAAV2 (WT) 1.5× 10¹² 4.6 × 10¹¹  1.8 × 10¹⁰ 25 + 3.4 H3558A <1.0 × 10⁸    <1.0 ×10⁶    <1.0 × 10⁴   N/D^(f) N/D N/D R447A 1.2 × 10¹² 3.4 × 10¹⁰ 1.3 ×10⁹ 25 + 35.9 R459A 9.1 × 10¹⁰ 7.2 × 10⁸  <1.0 × 10⁴   >72500 + 126.3R484A 1.5 × 10¹¹ 3.0 × 10¹⁰ <1.0 × 10⁴   >2976667 +/− 5.1 R487A 5.4 ×10¹¹ 2.2 × 10¹¹ 2.3 × 10⁸ 954 +/− 2.5 H509A 4.6 × 10¹⁰ 2.3 × 10⁹  6.9 ×10⁵ 3285 + 20.3 R513A 2.9 × 10¹¹ 1.7 × 10¹⁰ 1.6 × 10⁸ 106 + 17.9 K532A1.1 × 10¹¹ 3.6 × 10¹⁰ <1.0 × 10⁴   >3633333 +/− 3.0 K544A 2.0 × 10¹¹ 1.7× 10¹⁰ 8.3 × 10⁸ 20 + 11.9 R566A 5.1 × 10¹¹ 1.6 × 10¹⁰ 7.4 × 10⁸ 21 +32.6 R585A 5.0 × 10¹¹ 4.8 × 10¹⁰ 1.7 × 10⁷ 2812 − 1.4 R587A 4.4 × 10¹¹1.3 × 10¹⁰ 1.7 × 10⁷ 165 + 34.7 R588A 2.4 × 10¹¹ 5.6 × 10¹⁰ 3.0 × 10⁶18521 − 4.2 H526A, K527A 1.4 × 10¹¹ 8.2 × 10¹⁰ 5.5 × 10⁷ 1489 + 1.8R585A, R588A 1.2 × 10¹² 9.2 × 10¹¹ 1.9 × 10⁷ 48421 − 1.2 R585K 1.3 ×10¹² 3.7 × 10¹⁰ 4.0 × 10⁸ 92 + 35.4 R585K, R588K 1.4 × 10¹² 3.9 × 10¹⁰8.9 × 10⁷ 436 + 34.9 AAV1 N/D 3.7 × 10¹⁰ 1.1 × 10⁹ 37 +/− N/D AAV5 N/D3.4 × 10¹⁰ 3.2 × 10⁶ 10692 − N/D AAV5-HS N/D 8.0 × 10⁸  <1.0 ×10⁴   >80000 + N/D ^(a)Two letters flanking a number designate eachmutant. The first letter is the one letter abbreviation for the wildtype amino acid followed by its numerical position in VP1 followed bythe one letter abbreviation for the amino acid to which it was mutated.^(b)A20 particle titers were determined as described using the A20 ELISAassay. Genomic titers were determined by RT-PCR ™. ^(c)Infectious titerswere determined by green cell assay as described by counting GFPfluorescent cells. ^(d)Particle-to-infectivity ratio was calculated bydividing the average genomic titer as determined by RT-PCR ™ by theaverage green cell assay titer. ^(e)Determined by heparin-agarosebinding assay. +, >95% virus recovered in the eluate; +/−, >50 recoveredin the eluate; −, <5% of virus recovered in the eluate. ^(f)N/D, notdetermined. ^(g)Empty-to-full ratio was determined by dividing the A20particle titer by the average genomic titer.

To determine whether any mutations affected DNA packaging, the titer ofDNA containing virions was determined by real-time (RT) PCR™ (Clark etal., 1999; Veldwijk et al., 2002) (Table 5) and confirmed by DNA dotblot hybridization. Although there was variation between preparations,the majority of the capsid mutants were able to package detectable DNA(Table 5). As expected, H358A was negative for DNA packaging, as it didnot produce virus particles. It was concluded that none of the capsidsin the mutant panel that made A20 positive particles were completelydefective for DNA packaging. However, by comparing the A20 ELISA and PCRtiters, it was noted that stocks of mutant R459A contained 40-fold moreempty particles than wild type rAAV2. Thus, R459 could have a role inDNA packaging. Although less dramatic, mutants R447A, R566A, R587A,R585K, and R585K/R588K had approximately 10-fold more empty particlesthan rAAV2. The remainder of the virus preparations packaged DNA atlevels comparable to wild type AAV2 (Table 5).

In Vitro Heparin Binding of Capsid Mutants

To assess the ability of mutant capsids to bind heparin sulfate, amodification of an assay previously described by Wu et al. (2002) wasused. Virus preparations that had been purified by iodixanol stepgradients were loaded on heparin agarose columns and the entire volumeof the flow through, wash, and eluate fractions were pooled separately,denatured, and slot blotted onto nitrocellulose for immunoblot analysiswith B1 antibody. A representative Western analysis for each mutant isshown in FIG. 8. As expected, wild type AAV2 was not observed in theflow through or wash fractions and most of the virus bound to the columnwas recovered at the elution step. Eight other mutants, R447A, R459A,H5009A, R513A, K544A, K566A, N587A, and H526A/K527A, had aheparin-agarose binding phenotype indistinguishable from wild type. Theresults with R513A confirmed a previous report (Wu et al., 2000) inwhich a double mutant at positions 513 and 514 was positive for heparinbinding. In marked contrast, it was observed that any capsid harboring anon-conservative mutation at position 585 or 588 was detected only inthe flow-through and in the wash. Intermediate heparin-agarose bindingphenotypes in mutants R484A, R487A and K532A were also detected withapproximately equal levels of signal detected in the flow through, wash,and eluate. The results with K532A were inconsistent with previousresults in which a mutant containing alanine substitutions at positions527 to 532 was found to be positive for heparin binding (Wu et al.,2000). These data suggested that at least five amino acids had thepotential to contribute to the electrostatic attraction between AAV andheparin sulfate. These included predominantly R585 and R588, and to alesser but detectable extent, R484, R487, and K532.

To confirm that the charge at R585 and R588 was primarily responsiblefor heparin interaction, two viruses were generated with conservativemutations, R585K and R585K/R588K, and tested them in the in vitroheparin binding assay. Both lysine and arginine residues are positivelycharged, however, lysine is slightly larger due to an additional methylresidue in the side group. Both of these capsids bound toheparin-agarose almost as well as wild type virus (FIG. 8). In eachcase, most of the virus was recovered in the eluate; however, the flowthrough and wash fractions also contained minor amounts of virus. Thisresult suggested that both localized negative surface charge and therelative position of the changes in this region of the capsid wereresponsible for mediating the interaction with heparin-agarose.

Finally, as a control and to validate the heparin binding assay, theability of wild type rAAV2, rAAV1, and rAAV5 to bind to heparin-agarosewas compared. For this purpose, recombinant viruses were produced andpurified by using a pseudotyping protocol developed to package AAV2terminal repeat containing genomes into alternative serotype capsids(FIG. 9A) (Rabinowitz et al., 2002; Zolotukhin et al., 2002).Approximately equal amounts of input virus as determined by Western blotsignal intensity were applied to a heparin-agarose column, and fractionsfrom the column were slot blotted onto nitrocellulose forimmunodetection using the B1 antibody (FIG. 9B). As expected, rAAV2 wasefficiently retained by heparin-agarose under low ionic conditions butthe majority of rAAV1 and all of rAAV5 was seen in the flow through andwash. A low amount of AAV1 was detected in the eluate. These data wereconsistent with previous reports (Rabinowitz et al., 2002).

Multiple Mutations in the Aav2 Capsid Effect Viral Transduction

To determine how the heparin-agarose binding phenotypes correlated toinfectivity, iodixanol stocks were tested for their ability to transduceHeLa C12 cells by performing a green cell assay (GCA). Cells in a96-well plate were co-infected with Ad5 at a constant MOI=10 pfu/celland mutant AAV virus stocks in a 10-fold dilution series. Twenty-fourhrs post-infection (hpi), the number of GFP expressing cells inindividual wells were counted and a GCA titer was calculated (Table 5).The detection limit of this assay was approximately 10⁴ transducingunits/mL. The GCA titers were then normalized to genome containingphysical particles by calculating a particle-to-infectivity (P/I) ratio.This ratio is equivalent to the number of genomes required to transduceone cell (Table 5). To get a measure of the relative impact of aparticular mutation on viral infectivity, the P/I ratio of each mutantwas divided by the wild type capsid P/I ratio and the log_(in) of thisvalue was plotted in FIG. 10. This provided a simple comparison of howmany genome-containing particles of each mutant were required to achievethe same number of transduced cells as the wild type virus.

Several phenotypes emerged from this analysis. Mutants R477A, K544A, andK566A were virtually identical to wild type, and mutants R513A, N587A,R585K, and R585K/R588K were only slightly defective (approximately 1log). These seven mutants were found previously to bind heparin sulfateto the same extent as wild type rAAV2 (FIG. 8).

Three of the mutants R459A, R484A, and K532A produced virus that wasessentially non-infectious with P/I ratio between 7.2×10⁴ and 3.6×10⁶compared to the wild type ratio of 25 (Table 5, FIG. 10). The P/I ratiosfor these mutants were minimum estimates based on the GCA assaysensitivity of 1×10⁴ IU/mL. In fact, no transduction events were seenwith any of these mutants. R459A was the most severe example of threemutants (R459A, H509A, and H526A/K527A) that were essentially wild typefor heparin binding but defective for transduction (FIG. 10). Thesemutants were presumably defective in some late stage of viral infection.

Finally, all five of the mutants that were defective or partiallydefective for heparin binding (R484A, R487A, K532A, R585A, and R588A)were defective for transduction. However, the loss of infectivity didnot correlate completely with the loss of heparin binding (compare FIG.8 and FIG. 10). Two of these mutants (R484A and K532A) were onlypartially defective for heparin binding but severely defective (>5 logs)for transduction, suggesting that some other step in viral infection wasdefective in these mutants in addition to heparin binding. The remainingheparin binding mutants (R487A, R585A, and R588A) had defects intransduction that approximately correlated with their ability to bindheparin.

Evaluation of R585A/R588A Cell Attachment in Vivo

As mentioned earlier, alanine substitutions at either position 585 or588 were the only mutations that completely abolished binding to HS(FIG. 8), suggesting that these two arginines were primarily responsiblefor heparin binding. Moreover, the extent to which mutation of either orboth of these residues inhibited transduction (FIG. 10, 1.5-3 logs) wasapproximately the same when soluble heparin sulfate is used to inhibitwild type rAAV2 infection (Handa et al., 2000). Those mutants were,therefore, examined in more detail.

To see if the defect in transduction of R585 and R588 mutants could beovercome by using higher input MOI's, cells were co-infected with rAAV2or the mutant viruses at an MOI=500 genome containing particles/cell.Twenty-four hrs post-infection cells were examined by fluorescencemicroscopy and counted by FACS. The data from three independentexperiments and representative histograms are shown in FIG. 11. Asexpected, the defects in transduction of the single mutants, R585A andR588A, could be overcome by higher MOI's (56% and 25% transduction forR585A, and R588A, respectively). Predictably, the level of recovery ofthe double mutant, R585A/R588A, was lower (10% transduction). However,it was clear that the fluorescence intensity profile for the heparinbinding mutants was quite different from wild type, suggesting asignificant delay in the onset of GFP expression by 24 hrs. In contrast,the level of transduction of the conservative double mutant,R585K/R588K, and the heparin positive mutant, N587A, wasindistinguishable from wild type.

As a more direct assay for cell attachment, Hela C12 cells weretransfected and the location of viral DNA tracked. Cells were infectedwith rAAV2 at an MOI=100 or R585A/R588A at an MOI=1000 genome containingparticles as determined by RT-PCRTM. At 1, 4, and 20 hpi, the infectionmedia was removed and saved, and the cells were washed extensively toremove any residual unbound virus. The cells were then harvested and lowmolecular weight DNA was extracted from both the infection media(unbound) and the cell pellet (bound or internalized) by the Hirtprocedure and transferred to nitrocellulose for Southern hybridizationwith an [α-³²P]-dATP labeled GFP probe (FIG. 12A and FIG. 12B).

At all time points rAAV2 DNA was detectable both bound/internalized andin the infection media. In contrast, cells infected with 10-fold moregenomic copies of R585A/R588A showed the vast majority of the signalonly in the unbound fraction (FIG. 12A). Phosphor imager analysisdetermined that at each time point approximately one third of the totalrAAV2 DNA was attached or internalized compared to only 1% ofR585A/R588A (FIG. 12B). As these infections were performed at 37° C.,the process of internalization should not have been prevented. Thisresult demonstrated that the block in infection for mutant R585A/R588Aoccurred at the cell attachment stage or internalization stage, andcorrelated to heparin sulfate binding in vitro.

Loop Swapping Confers Heparin Binding to AAV5

Although the primary amino acid sequences are moderately divergent, thearchitectural position of β-sheets and loops is predicted to be verysimilar among AAV serotypes (Rabinowitz and Samulski, 2000). It washypothesized that if R585 and R588 were the critical residues involvedin HSPG binding, then it should be possible to substitute that region ofAAV2 into AAV5 to create a hybrid virus capable of interacting withheparin-agarose. To achieve this, a recombinant virus, designatedrAAV5-HS, was generated by replacing a short loop containing residues585 through 590 from AAV2 into a region predicted to be structurallyequivalent in AAV5 (FIG. 13A). Loop substitution rather than pointmutagenesis was done to account for the possibility of additional Vander Waals interactions or hydrophobic contributions from nearby aminoacids.

Production and purification of rAAV5-HS was unaffected by the six aminoacid substitution (FIG. 13B; Table 5). When rAAV5-HS was tested in thein vitro heparin-agarose binding assay, it was indistinguishable fromwild type rAAV2 (FIG. 13C). These data suggested that this region ofAAV5 was surface accessible, and that heparin-agarose binding could beartificially conferred by the six amino acids containing R585 and R588.

To compare the infectivity of rAAV5 and rAAV5-HS, packaged viruses weregenerated that contained a recombinant AAV5 genome in which the GFPreporter gene was flanked by AAV5 terminal repeats. The infectivity ofthese viruses was compared to rAAV2 in a GCA assay andparticle-to-infectivity ratios were calculated as before (FIG. 13D).rAAV5 was able to transduce Hela C12 cells at a low efficiency,approximately 2.5 logs lower than AAV2. However, no transduction wasseen with AAV5-HS (<1×10⁴ IU/mL) (Table 5; FIG. 13D). Given the minimumsensitivity of the GCA assay this meant that the P/I ratio of AAV5-HSwas at least 3.5 logs higher than rAAV2 and at least 1 log higher thanwild type rAAV5. It was concluded that, although substitution of thesefive heterologous amino acids into the AAV5 capsid restored heparinbinding to the level of AAV2 capsids, it was not sufficient to produceAAV2 levels of infectivity in a cell line normally permissive for AAV2.

Discussion

This example describes the identification of amino acids in the capsidof AAV2 that mediate binding to heparin sulfate proteoglycans. Severallines of evidence suggest that HSPG serves as the primary receptor forAAV2 Inhibition of AAV2 infection can be demonstrated by competitionwith soluble analogs, GAG desulfation by sodium chlorate treatment,antibody competition, enzymatic removal of heparin, and use of mutantcell lines that express varying levels of HSPG (Handa et al., 2000; Qiuet al., 2000; Summerford and Samulski, 1998; Wu et al., 2000). Bindingto heparin sulfate is usually the result of electrostatic chargeinteractions between basic amino acids (R, K, or H) and negativelycharged sulfate residues (Hileman et al., 1998; Mulloy and Linhardt,2001). During the course of previous mutagenesis studies, many of thebasic amino acids in the AAV2 capsid that could potentially contributeto heparin sulfate binding were eliminated (Wu et al., 2000). In thisexample, the remaining basic residues were examined by looking at theirconservation in AAV serotypes 1 to 5. Those that were present in allfive serotypes were not likely to contribute significantly to heparinbinding. Those that were conserved in the heparin binding serotypes,AAV1 to 3, but not in the remaining serotypes were targeted formutagenesis. Finally, by taking advantage of the fact that R585 and R588had been previously identified as potential heparin binding amino acids(Wu et al., 2000) and that these amino acids were located in a clusterof basic residues at the three fold axis of symmetry (Xie et al., 2002),all of the basic amino acids in this cluster were also targeted formutagenesis. This approach yielded a total of 15 amino acids that couldhave been involved in heparin binding and alanine mutations werecharacterized at all of these positions. This approach, of course, doesnot necessarily identify all possible heparin binding amino acids. Forexample, R484, which is basic in all five serotypes was tested becauseof its proximity to R585 and R588 and subsequently proved to be involvedin heparin binding.

Heparin Binding and Infectivity

These studies indicated that capsids with mutations at residue 484, 487,532, 585 or 588, were partially or completely defective forheparin-agarose binding. The most severe defect was found with mutationsin R585 and R588. No binding to heparin sulfate columns could bedetected with either mutant (FIG. 8), and both mutations reduced theparticle-to-infectivity ratio by two to three logs (Table 5). Mutantsthat contained substitutions at both positions had even lowerinfectivity.

The phenotypes of R487A, R585A, and R588A, were probably due largely todefective heparin binding. For example, the double mutant R585A/R588Awas approximately 500 fold more defective in cell binding andinternalization than wild type (FIG. 12B) when corrected for the MOI,and approximately 2000 fold less infectious (Table 5), as judged by thechange in particle-to-infectivity ratio. Another indication that heparinbinding was primarily responsible for the defects in R585 and R588 wasthe fact that conservative mutations at these two positions (R585K andR585K/R588K) produced virus particles with properties similar to wildtype (FIG. 8; FIG. 10; FIG. 11; Table 5). Results from the conservativelysine substitutions at R585 and R588 are reasonably consistent withelectrostatic attraction being the primary mediator for AAV-heparininteraction. R585K, the least defective heparin binding mutant (FIG. 8),had transduction levels nearly equal to rAAV2 (FIGS. 10), andR585K/R588K was only slightly more defective for heparin binding (FIG.8) and transduction (FIG. 10), and within one log of wild type.Furthermore, when cells were infected at a high MOI, robust transductionwas observed for both mutants (FIG. 11). Finally, substitution of a sixamino acid sequence containing R585 and R588 imparted heparin binding toAAV5 that was comparable to that seen with AAV2 (FIG. 13). Althoughsimilar studies were not performed with the R487 position, it was clearthat mutation of R487 produced virus with a more modest defect inheparin binding (FIG. 8) and in infectivity (FIG. 10).

In addition to R487A, R585, and R588, two other mutants were found thatwere defective for heparin binding, R484A and K532A. R484A and K532A,like R487A, had more modest effects on binding to heparin sulfate, butunlike the other heparin binding mutants, these two mutations had adramatic effect on transduction efficiency. Both R484A and R532A weremore than 5 logs less infectious than wild type capsids (Table 5; FIG.10). This severe defect is presumably due to a different block in theinfection process that is unrelated to heparin binding, but as yet ithas not been identified. The result from K532A is consistent withearlier studies that identified a mutant (mut37) that contained sixamino acid substitutions that included K532A (Wu et al., 2000). Mut37had a phenotype identical to K532A in that it produced full virusparticles that were non-infectious and more recently has been shown tohave a modest defect (approximately 5-fold) in heparin binding andinternalization. This potentially maps this defect to a single aminoacid.

Computer Modeling

Using the recently published atomic structure of AAV2 (PDB ID code:ILP3) (Xie et al., 2002), the positions of the heparin binding mutationswere examined. Symmetry transformation operations from the original PDBfile were applied to generate a VP3 trimer arrangement in the context ofan icosahedron. When viewed in ribbon format looking directly down athree-fold axis, residues R484, R487, R532, R585 and R588, representedas balls-and-sticks, were located in a linear formation lining one sideof each three-fold related spike. When viewed across the top surface ofthe trimer, residues R585 and R588, which are contributed by one of thepeptides in the trimer, were positioned above a linear arrangement ofR484, R487 and K532, which are contributed by a second peptide in thetrimer. Thus, it appears that a heparin binding motif is formed fromsome combination of these five amino acids using amino acids from twodifferent polypeptides. An electrostatic potential surface map of a VP3trimer was also generated, in which areas of positive and negativecharge are represented. When viewed perpendicular to the three-foldaxis, the five amino acids mapped by this example appear to contributecollectively to a basic patch on one side of each three-fold-relatedspike. The charge, clustering, and surface presentation of theseresidues were all consistent with a model of electrostatic attraction.Two other basic residues, H526 and K527, contribute to the basic clusterat the three fold spike but these residues do not appear to be involvedin heparin binding (FIG. 8).

The five mutations that affected heparin binding were located in thelarge loop IV region, which among AAV serotypes has low overall sequenceconservation and includes all of the previously identified insertion andsubstitution mutations that affect heparin binding. Interestingly, withthe exception of N587, the stretch of amino acids encompassing 585 to590 is unique to AAV2 and is not present in AAV3, which is the other AAVserotype that has been shown to bind efficiently to heparin sulfate.Mutation of N587 had no effect on heparin-agarose binding and only minoreffects on transduction. Conceivably, residues R484, R487, and K532could be the dominant residues involved in heparin sulfate binding forAAV3.

The apparent dissociation constant (K_(d)) of AAV2 and heparin sulfatewas determined by competition analysis to be 2×10⁹ M (Qiu et al., 2000).Although this is higher than some heparin-protein interactions, it issufficiently strong to suggest cooperative binding by one HSglycosaminoglycan chain to multiple attachment points. This example doesnot address whether heparin sulfate could form a bridge between basicresidues in one of the threefold spikes to those in another. However, asthe average chain length of heparin glycosaminoglycans varies between 50and 200 disaccharide repeats that adopt a helical conformation 40-160 nmin length, it is conceivable that a heparin sulfate chain could wraparound the exterior of the capsid through cooperative binding ofmultiple spikes at the threefold axis of symmetry. Although a rigorouscomputational docking analysis was not undertaken, a heparin molecule(PDB ID code 1NTP) was manually superimposed in several orientationsthat placed multiple reactive sulfate and amine groups within acceptedelectrostatic attraction distances on pairs of residues spanning thespikes.

Mutants that Bind Heparin but are Still Defective

Several new mutants were found that bound heparin sulfate as well aswild type but still produced defective particles. H538A was defectivefor particle assembly. There are a number of reported examples ofmutations that disrupt AAV2 particle formation, several of which arelocated in the conserved β-strand regions (Rabinowitz et al., 1999; Shiet al., 2001; Wu et al., 2000). Since H358 is neither surface accessiblenor in a conserved β-strand, it is possible that it acts internally tostabilize the monomer subunit structure.

Mutants R459A, H509A, and H526A/K527 bound heparin-agarose efficientlybut had particle-to-infectivity ratios that were two to more than threelogs higher than wild type. Like K532A and R484A, these mutants arepresumably defective in some stage of the infectious entry pathwaybetween secondary receptor binding and uncoating. Ongoing studies in thelab are examining the block in infectivity for these mutants.

DNA Packaging

The process of DNA packaging is thought to occur by an active processrequiring NTP consumption coupled to the helicase activity of the smallRep proteins (King et al., 2001). Although none of the mutations thatassembled an A20-positive particle were completely deficient for DNApackaging, mutant R459A produced a 40-fold excess of empty capsidparticles compared to rAAV2. Other studies have reported that shortinsertions at positions 323, 339, 466, 520, 540, 595, and 597 that didnot interfere with capsid formation still reduced DNA packaging tolevels detectable only by PCR™ amplification (Shi et al., 2001). Inaddition, a point mutant, R432A, prevented DNA packaging (Wu et al.,2000). Although the relationship between these mutations and theirmechanism of action is unclear, it is possible that they disruptprotein-capsid or DNA-capsid interactions.

Summary of Exemplary Production System

An exemplary rAAV production system has been described to producemodified rAAV vectors that comprise one or more altered capsid proteins.FIG. 14 shows the results of an immunoslotblot of total capsid proteinfollowing standard purification procedures of a representativeexpression system of the invention. FIG. 15 shows a dot blotautoradiograph of DNA extracted from pTR-UF5 and the system plasmidcombinations. FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D show the invivo transduction ability of recombinant AAV vectors produced usingvarious system components. FIG. 17A and FIG. 17B show an Immunoblot anddot blot autoradiograph of virions produced from pTR-UF5; pIM45-VP1,2;pIM45-VP1,3; and pIM45-VP2,3 plasmids following standard purificationprotocols. FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show the in vivotransduction ability of recombinant AAV vectors containing only twocapsid proteins, while FIG. 19 depicts an immunoblot of proteinfractions collected from iodixinol purified passed over aheparin-agarose column. Using an anti-VP1,2,3 monoclonal antibody, FIG.20 shows a dot blot autoradiograph of DNA extracted from pTR-UF5 andrAAV R585A, R588A, while FIG. 21A, FIG. 21B, FIG. 21C, and FIG. 21Dsummarize an exemplary system that demonstrates the in vivo transductionability of pTR-UF5 and R585A, R588A.

FIG. 22 shows a slot blot autoradiograph of an in vivo DNA tracking timecourse experiment of pTR-UF5, rAAV R585A, R588A, while FIG. 23 shows aschematic diagram of the pIM45 vector showing the rep and cap sequences.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein in their entirety by express referencethereto:

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

What is claimed is:
 1. A method for targeting a selected therapeuticagent to a mammalian cell, the method comprising providing to the cellan effective amount of a composition comprising: (a) an rAAV vectorsystem that comprises or that encodes a selected therapeutic agent, andthat further comprises: (i) a first expression vector comprising acoding sequence of a first AAV capsid protein under control of anexpression control sequence; and (ii) a second expression vectorcomprising coding sequences for a second AAV capsid protein undercontrol of an expression control sequence and a third AAV capsid proteinunder control of an expression control sequence; wherein the first andthe second expression vectors are not on the same nucleic acid segment,and wherein the first, the second, and the third capsid proteins aredistinct capsid proteins selected from the group consisting of an AAV2Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and furtherwherein the composition is modified 1) by a mutation in the codingsequence for the Vp2 protein such that the coding sequence for Vp2 doesnot express a functional AAV Vp2 capsid protein and/or 2) by at leastone mutation such that binding to HPSG is altered, impaired, orprevented, wherein the at least one mutation is an arginine-to-alaninemutation, or an arginine-to-lysine mutation at an amino acid residuecorresponding to R487, R585, or R588 of an AAV2 capsid protein; or (b)an infectious rAAV virion that comprises or that encodes a selectedtherapeutic agent, and that further comprises: (i) a first expressionvector comprising a coding sequence of a first AAV capsid protein undercontrol of an expression control sequence; and (ii) a second expressionvector comprising coding sequences for a second AAV capsid protein undercontrol of an expression control sequence, and a third AAV capsidprotein under control of an expression control sequence; wherein thefirst and the second expression vectors are not on the same nucleic acidsegment, and wherein the first, the second, and the third capsidproteins are distinct capsid proteins selected from the group consistingof an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, andfurther wherein the composition is modified 1) by a mutation in thecoding sequence for the Vp2 protein such that the coding sequence forVp2 does not express a functional AAV Vp2 capsid protein and/or 2) by atleast one mutation such that binding to HPSG is altered, impaired, orprevented, wherein the at least one mutation is an arginine-to-alaninemutation, or an arginine-to-lysine mutation at an amino acid residuecorresponding to R487, R585, or R588 of an AAV2 capsid protein.
 2. Themethod of claim 1, wherein the selected therapeutic agent comprises aribozyme, an antisense molecule, a peptide, a polypeptide, a protein, anantibody or an antigen binding fragment thereof, or any combinationthereof.
 3. The method of claim 2, wherein the selected therapeuticagent is a protein or a polypeptide selected from the group consistingof an adrenergic agonist, an anti-apoptosis factor, an apoptosisinhibitor, a cytokine receptor, a cytokine, a cytotoxin, anerythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, agrowth factor, a growth factor receptor, a hormone, a hormone receptor,an interferon, an interleukin, an interleukin receptor, a kinase, akinase inhibitor, a nerve growth factor, a netrin, a neuroactivepeptide, a neuroactive peptide receptor, a neurogenic factor, aneurogenic factor receptor, a neuropilin, a neurotrophic factor, aneurotrophin, a neurotrophin receptor, an N-methyl-D-aspartateantagonist, a plexin, a protease, a protease inhibitor, a proteindecarboxylase, a protein kinase, a protein kinase inhibitor, aproteolytic protein, a proteolytic protein inhibitor, a semaphorin, asemaphorin receptor, a serotonin transport protein, a serotonin uptakeinhibitor, a serotonin receptor, a serpin, a serpin receptor, and atumor suppressor.
 4. The method of claim 3, wherein the protein orpolypeptide is selected from the group consisting of BDNF, CNTF, CSF,EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF,PEDF, TGF, TGF-B2, TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A),viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.5. The method of claim 1, wherein the mammalian cell is a human, aprimate, a murine, a feline, a canine, a porcine, an ovine, a bovine, anequine, an epine, a caprine, or a lupine cell.
 6. The method of claim 5,wherein the mammalian cell is a human endothelial cell, a human vascularcell, a human epithelial cell, a human liver cell, a human lung cell, ahuman cardiac cell, a human pancreatic cell, a human renal cell, a humanmuscle cell, a human bone cell, a human neural cell, a human blood cell,or a human brain cell.
 7. The method of claim 1, wherein the compositionfurther comprises a liposome, a lipid, a lipid complex, a microsphere, amicroparticle, a nanosphere, a nanoparticle, or any combination thereof.8. A method for treating or ameliorating one or more symptoms of adisease, a dysfunction, or a deficiency in a mammal, the methodcomprising: administering to a mammal in need thereof, an effectiveamount of a composition comprising: (a) an rAAV vector system thatcomprises or that encodes a selected therapeutic agent, and that furthercomprises: (i) a first expression vector comprising a coding sequence ofa first AAV capsid protein under control of an expression controlsequence; and (ii) a second expression vector comprising codingsequences for a second AAV capsid protein under control of an expressioncontrol sequence and a third AAV capsid protein under control of anexpression control sequence; wherein the first and the second expressionvectors are not on the same nucleic acid segment, and wherein the first,the second, and the third capsid proteins are distinct capsid proteinsselected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2protein and an AAV2 Vp3 protein, and further wherein the composition ismodified 1) by a mutation in the coding sequence for the Vp2 proteinsuch that the coding sequence for Vp2 does not express a functional AAVVp2 capsid protein and/or 2) by at least one mutation such that bindingto HPSG is altered, impaired, or prevented, wherein the at least onemutation is an arginine-to-alanine mutation, or an arginine-to-lysinemutation at an amino acid residue corresponding to R487, R585, or R588of an AAV2 capsid protein; or (b) an infectious rAAV virion thatcomprises or that encodes a selected therapeutic agent, and that furthercomprises: (i) a first expression vector comprising a coding sequence ofa first AAV capsid protein under control of an expression controlsequence; and (ii) a second expression vector comprising codingsequences for a second AAV capsid protein under control of an expressioncontrol sequence, and a third AAV capsid protein under control of anexpression control sequence; wherein the first and the second expressionvectors are not on the same nucleic acid segment, and wherein the first,the second, and the third capsid proteins are distinct capsid proteinsselected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2protein and an AAV2 Vp3 protein, and further wherein the composition ismodified 1) by a mutation in the coding sequence for the Vp2 proteinsuch that the coding sequence for Vp2 does not express a functional AAVVp2 capsid protein and/or 2) by at least one mutation such that bindingto HPSG is altered, impaired, or prevented, wherein the at least onemutation is an arginine-to-alanine mutation, or an arginine-to-lysinemutation at an amino acid residue corresponding to R487, R585, or R588of an AAV2 capsid protein; in an amount and for a time sufficient totreat or to ameliorate the one or more symptoms of the disease, thedysfunction, or the deficiency in the mammal.
 9. The method of claim 8,wherein the composition is administered to the mammal intra-muscularly,intravenously, subcutaneously, intrathecally, intraperitoneally, or bydirect injection into one or more organs or tissues of the mammal. 10.The method of claim 9, wherein the one or more organs or tissues areselected from the group consisting of pancreas, liver, heart, lung,brain, kidney, joint, and muscle.
 11. The method of claim 8, wherein therAAV vector system or the infectious rAAV virion further comprises athird distinct expression vector that comprises an expression cassetteflanked by AAV2 terminal repeat sequences.
 12. The method of claim 11,wherein the expression cassette comprises a first polynucleotide thatcomprises a first nucleic acid segment that encodes a selectedtherapeutic agent.
 13. The method of claim 12, wherein the selectedtherapeutic agent is a peptide, a polypeptide, a protein, a catalyticRNA molecule, a ribozyme, an antisense oligonucleotide, or an antisensepolynucleotide.
 14. The method of claim 12, wherein the firstpolynucleotide further comprises a promoter operably linked to the firstnucleic acid segment, wherein the promoter controls expression of theselected therapeutic agent in a mammalian cell.
 15. The method of claim14, wherein the promoter is a heterologous promoter, a tissue-specificpromoter, a constitutive promoter, or an inducible promoter.
 16. Themethod of claim 12, wherein the first polynucleotide further comprisesan enhancer sequence operably linked to the first nucleic acid segment.17. The method of claim 16, wherein the enhancer sequence comprises aCMV enhancer, a synthetic enhancer, a liver-specific enhancer, anvascular-specific enhancer, a brain-specific enhancer, a neuralcell-specific enhancer, a lung-specific enhancer, a muscle-specificenhancer, a kidney-specific enhancer, a pancreas-specific enhancer, oran islet cell-specific enhancer.
 18. The method of claim 12, wherein thefirst polynucleotide further comprises a post-transcriptional regulatorysequence or a polyadenylation signal operably linked to the firstnucleic acid segment.
 19. The method of claim 18, wherein thepost-transcriptional regulatory sequence is obtained from a woodchuckhepatitis virus post-transcription regulatory element, or wherein thepolyadenylation signal is obtained from a bovine growth hormone gene.20. A method for targeting an AAV virion or viral particle to amammalian cell that comprises a cell-surface receptor, the methodcomprising: providing to a population of mammalian cells a recombinantadeno-associated viral expression system comprising: (i) a firstexpression vector comprising a coding sequence of a first AAV capsidprotein under control of an expression control sequence; and (ii) asecond expression vector comprising coding sequences for a second AAVcapsid protein under control of an expression control sequence and athird AAV capsid protein under control of an expression controlsequence; wherein the first and the second expression vectors are not onthe same nucleic acid segment, and wherein the first, the second, andthe third capsid proteins are distinct capsid proteins selected from thegroup consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2Vp3 protein, and further wherein the composition is modified 1) by amutation in the coding sequence for the Vp2 protein such that the codingsequence for Vp2 does not express a functional AAV Vp2 capsid proteinand/or 2) by at least one mutation such that binding to HPSG is altered,impaired, or prevented, wherein the at least one mutation is anarginine-to-alanine mutation, or an arginine-to-lysine mutation at anamino acid residue corresponding to R487, R585, or R588 of an AAV2capsid protein; in an amount and for a time effective to target thevirion or the viral particle to one or more cells of the population thatexpress the cell-surface receptor.
 21. The method of claim 20, whereinthe rAAV vector system further comprises a third distinct expressionvector that comprises an expression cassette flanked by AAV2 terminalrepeat sequences, wherein the expression cassette comprises a firstpolynucleotide that comprises a first nucleic acid segment that encodesa selected therapeutic agent operably linked to a promoter that controlsexpression of the selected therapeutic agent in a mammalian cell. 22.The method of claim 21, wherein the selected therapeutic agent is apeptide, a polypeptide, a protein, a catalytic RNA molecule, a ribozyme,an antisense oligonucleotide, or an antisense polynucleotide.